Transcriptional Regulation of One-Carbon Metabolism Genes of Saccharomyces cerevisiae
A thesis presented for the degree of Doctor of Philosophy by Seung-Pyo Hong
School of Biochemistry and Molecular Genetics
University of New South Wales, Australia
1999 CERTIFICATE OF ORIGINALITY
I hereby declare that this submission is my own WOJ:k and to the best of my knowledge it contains no materials previously published or wriacn by another person, nor material which to a substanti21 extent has been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due: acknowledgc:me:nt is made in the thesis. Any coatnlmtion made to the research by other.;, with whom I have WOiked at UNSW or elsewhere, is expliciliy acknowledged in the thesis.
I also declare that the intellectual content of this thesis is 1bc product of my own work, except 10 the extent that assistance from others in the project's design and conception or in style., ~on and I:i:nguisti.c expression is acknowledged
(S;gned) .. ~-· ...... TABLE OF CONTENTS
Summary ...... i Publications ...... iv Abbreviations ...... v Acknowledgments ...... vi
CHAPTER 1: INTRODUCTION
1.1 Overview ...... !
1.2 One-Carbon Metabolism ...... 2
1.2.1 Tetrahydrofolate, a carrier molecule of one-carbon metabolism ...... 2
Clinical importance of one-carbon metabolism and THF inhibitors ..... 3
The role of polyglutamate tails of THF derivatives ...... 4
1.2.2 Donors of one-carbon units in S. cerevisiae ...... 6
Major one-carbon donors in yeast...... 6
Source of one-carbon donors in S. cerevisiae ...... 8
1.2.3 One-carbon flux in S. cerevisiae ...... 9
One-carbon flux through SHMT isozymes ...... 10
Cytoplasmic and mitochondrial one-carbon flux ...... lO
1.2.4 Compartmentation of one-carbon metabolites: a proposed model. .... l2
Substrate channelling ...... l2
Implication of microcompartmentation inS. cerevisiae ...... l2
1.2.5 Genetic studies of one-carbon metabolism inS. cerevisiae ...... 14
Utilisation of glycine by mitochondria...... l4
Role of the C1-THF synthase in one-carbon metabolism ...... IS
1.2.6 Regulation of one-carbon metabolism ...... l7
1.3 Glycine Decarboxylase Multi enzyme Complex (GDC) ...... 18
1.3.1 The role of GDC ...... l8 The P-protein of GDC ...... 20
The H-protein of GDC ...... 20
The T -protein of GDC ...... 21
The L-protein of GDC ...... 22
1.3.2 Regulation of GDC in E. coli ...... 23
1.4 Regulation of Nitrogen Metabolism in S. cerevisiae ...... 25
1.4.1 Introduction ...... 25
1.4.2 Nitrogen catabolic repression (NCR) ...... 27
1.4.3 Nitrogen regulatory circuit in S. cerevisiae ...... 28
1.5 The Mechanism of Transcriptional Regulation ...... 33
1.5.1 RNA polymerase II general transcription machinery ...... 33
Stepwise assembly model of PIC ...... 34
RNA pol II holoenzyme ...... 37
1.5.2 Chromatin structure and transcription ...... 40
1.5.3 Relevant transcription factors ...... 43
General control of amino acid biosynthesis ...... 43
Bas lp/Bas2p ...... 4 7
1.6 Aims of the Thesis ...... 49
CHAPTER 2: MATERIALS AND METHODS
2.1 Materials ...... 51
2.1.1 General materials and reagents ...... Sl
2.1.2 Radiochemicals ...... Sl
2.1.3 Enzymes and related materials ...... 52
2.1.4 Oligonucleotides ...... 52
2.1.5 DNA and vectors ...... 54
2.1.6 Bacterial and S. cerevisiae strains ...... 55
Escherisclzia coli strains ...... 55 S. cerevisiae strains ...... 55
2.1.7 Media ...... 56
E. coli maintenance and growth media ...... 56
Saccharomyces cerevisiae media ...... 57
2.2 General Procedures ...... 57
2.2.1 Sterilization and containment of biological material...... 57
2.2.2 Buffers and stock solutions ...... 58
2.2.3 Substrate and enzyme stock solutions ...... 59
2.2.4 Preparation of dialysis tubing ...... 60
2.3 DNA Manipulations ...... 60
2.3.1 Restriction digestion of DNA ...... 60
2.3.2 Blunt end generation ...... 60
2.3.3 Phosphorylation of synthetic oligonucleotides ...... 61
2.3.4 Insertion of small DNA fragment...... 61
2.3.5 Ligation of DNA ...... 61
2.3.6 Site-directed mutagenesis ...... 62
2.4 Preparation of DNA and RNA ...... 62
2.4.1 Plasmid DNA preparation and purification ...... 62
Small scale plasmid preparation ...... 62
Midi-scale preparation ...... 63
Polyethylene glycol (PEG) precipitation ...... 63
CsCI density gradient centrifugation ...... 63
Phenol extraction ...... 63
Ethanol precipitation ...... 64
2.4.2 Yeast genomic DNA preparation ...... 64
2.4.3 RNA preparation ...... 64
Yeast total RNA preparation ...... 64
mRNA preparation ...... 65
2.4.4 Estimation of DNA and RNA concentration and purity ...... 65 2.4.5 Sequencing ...... 65
2.5 Cell Manipulations ...... 65
2.5.1 General yeast genetic techniques ...... 65
2.5.2 Transformations and transfections ...... 65
E. coli transformation ...... 66
S. cerevisiae transformation ...... 66
2.5.3 Yeast deletion mutant generation ...... 66
2.5.4 Yeast petite mutant generation ...... 67
2.6 Gel Electrophoresis ...... 67
2.6.1 Agarose gel electophoresis ...... 67
2.6.2 Polyacrylamide gel electophoresis ...... 67
2. 7 Radioactive Labelling and Autoradiography ...... 68
2.7 .1 End labelling ...... 68
2. 7.2 Random hexamer labelling ...... 68
2.7.3 eDNA labelling ...... 69
2.7.4 Southern analysis ...... 69
2.7 .5 Autoradiography and Photography ...... 69
2.7.6 Gene-array analysis ...... 70
2.8 Protein-DNA Interaction Study ...... 70
2.8.1 Yeast protein extract preparation ...... 70
Nuclear protein extraction ...... 70
Protein preparation by heparin-Sepharose chromatography ...... 71
2.8.2 Eletrophoretic Mobility Shift Assay (EMSA) ...... 72
2.8.3 Footprinting analysis ...... 72
Cu2+Jphenanthroline footprinting ...... 72
DNasel footprinting Analysis ...... 73
2.9 6-Galactosidase Assay ...... 7 4
2.10 Computer Analysis ...... 75 CHAPTER 3: ANALYSIS OF THE REGULATION OF GCV GENES IN VIVO
3.1 Regulation of GCV Genes by Different Nutrients ...... 76
3.1.1 Introduction ...... 76
3.1.2 Structure of promoter region of GCV genes ...... 77
3.1.3 Regulation of the GCV genes inS. cerevisiae ...... 82
3.2 Promoter Analysis of GCV Genes ...... 89
3.2.1 Identification of two potential regulatory elements ...... 89
3.2.2 Delimination of GRR (glycine regulatory region) in GCV2 ...... 96
3.3 The Glycine Regulatory Region in Heterologous Promoters ... 103
3.3.1 The glycine response of GCVJ and GCV2 is mediated by
repression ...... 103
3.3.2 The glycine response can also be mediated by activation ...... 105
3.4 Expression Study of the GCV Genes in Various Mutants ...... 107
3.4.1 gcn4 and bas 1 mutants ...... 107
3.4.2 NCR-regulatory mutants ...... 112
3.5 Conclusion ...... 1 18
CHAPTER 4: DNA-PROTEIN-SIGNALLING MOLECULE INTERACTIONS
4.1 Electrophoretic Mobility Shift Assay (EMSA) ...... 120
4.1.1 Introduction ...... l20
4.1.2 DNA-binding studies with nuclear extracts ...... l20
4.1.3 Effect of in vitro addition of one-carbon metabolites in EMSA ...... l26
4.1.4 foil mutan\...... 132
4.1.5 DNA-binding studies with heparin-Sepharose fractions ...... 136
4.2 Footprinting Analysis ...... 141
4.2.1 lntroduction ...... l41 4.2 .2 ON asel footprinting ...... 141
4.2.3 OP-Cu ( 1,1 0-phenanthroline-copper) footprinting ...... 145
CHAPTER 5: GENOME-WIDE ANALYSIS OF ONE-CARBON METABOLISM
5.1 Introduction ...... 148
5.2 Genome-Wide Transcriptional Analysis ...... 150
5 .2.1 Methodology ...... 150
5.2.2 Functional sorting of transcriptome ...... 152
5 .2. 3 One-carbon metabolism ...... 153
One-carbon metabolic flow ...... 154
Identification of YER 183C ...... 155
5 .2.4 Global metabolic flow ...... 158
5 .2.5 Transcription -related genes ...... 159
5 .2. 6 Other functional groups ...... 161
5.3 Future Direction ...... 163
CHAPTER 6: GENERAL DISCUSSION AND PERSPECTIVES 6.1 One-Carbon Regulon ...... l65
6.2 Dual Acting Transcription Factors ...... l66
6.3 Model for the Regulation of GCV Genes ...... 174
6.4 The GRR of Other One-Carbon Metabolism-Related Genes ..... 179
6.5 Future Directions ...... 180
References ...... 182
Appendix ...... 2 14 Summary
The glycine decarboxylase complex (GDC) of Saccharomyces cerevisiae is composed of four subunits (P, H, T and L) and plays an important role in the interconversion of serine and glycine and balancing the one-carbon unit requirements of the cell. It also enables the cell to use glycine as sole nitrogen source. This study was concerned with characterising the molecular mechanism of transcriptional regulation of the GCV genes encoding the subunits of the GDC. The important findings of this work can be summarised as follows: i) Transcription of the GCV genes are regulated by glycine and rich nitrogen sources, which are mediated by different cis-acting elements. The LPDI gene did not show a glycine response since its transcriptional regulation is distinct from that of the other genes encoding the GDC subunits. ii) Glycine analogues or serine did not affect expression of GCV2, and therefore glycine probably needs to be metabolised to effect the glycine response of the GCV genes. iii) The repression of the GCV2 gene expression by rich nitrogen sources is mediated by a sequence between -227 and -205 of GCV2, and NCR-regulatory mutant studies showed that repression is not directly controlled by the known NCR system. iv) The glycine response of GCV2 is mediated by a motif (the glycine regulatory region; GRR; 5'-CATCN7CTTCTT-3') with CTTCTT at its core. Additional sequence immediately 5' of this motif (between -310 to -289) plays a minor role for the gene's full glycine response. v) The GRR of the GCV genes can mediate the glycine response by either activation or repression, indicating that the transcription factor(s) mediating the glycine response is/are dual-functional in nature. vi) Studies of GCV2 gene expression using different regulatory mutants showed that expression of the gene is further modulated by other transcription factors such as Gcn4p and Bas I p which are distinct from the glycine response and possibly involved in setting up the basal expression level.
1 vii) In vitro studies of the GRR-protein interaction revealed THF affects the affinity
of the DNA-binding protein(s) for the GRR. The importance of THF in regulation of the
CCV2 gene was also shown in vivo using a foil mutant that is unable to synthesise any
folates. THF or a C 1-bound derivative of it acts as a ligand for the transcription factor,
thus influencing transcription of the CCV genes in the appropriate physiological manner.
viii) Using heparin-Sepharose chromatography fractions, four complex formations
(complex I to IV) were observed with the GRR. The protein responsible for one of these
was separable from the others. EMSA profiles using the GRR of the CCVI and CCV2
genes (in the presence or absence of THF) were very similar, indicating that these genes
bind the same proteins and are regulated in a similar manner.
ix) Mutation of the CTTCTT motif within the GRR caused significant reduction in in
vitro DNA-protein complex formation, however, THF addition overcame this reduction.
x) Only complex II formation was observed with a DNA fragment spanning -322 to
-295, and THF affected this complex formation.
xi) Footprinting analyses of complex I revealed that the binding protein protected the
GRR of the CCV2 gene from DNasei activity. This protein is an excellent candidate for
the glycine response regulatory protein. Titration experiments using EMSA showed that
this protein can dimerise.
A preliminary genome-wide analysis of the S. cerevisiae transcriptome was
carried out using miniarray membrane hybridisation. This investigated the global
transcriptional changes within the cell in response to the addition of glycine into the
medium. Identification of genes related to various cellular processes including one
carbon metabolism gave an insight into the regulation of the cellular metabolic flow, especially that of one-carbon metabolism. The results indicated that:
xii) Glycine is transported into mitochondria to be used as substrate for the GDC
which (with mitochondrial SHMT) produces serine that is subsequently utilised for the
various one-carbon metabolic pathways, such as methionine synthesis and purine
synthesis.
11 xiii) A gene of unknown function (YER 183C) which showed homology to the gene for human 5,10-CH-THF synthetase was identified from gene-array analysis to be up regulated on glycine addition, indicating the protein encoded by this gene may be involved in balancing the metabolic flow between methionine and purine synthesis when
THF pools are disturbed by glycine addition. xiv) Addition of glycine to the medium also triggers the expression of other metabolic genes related to amino acid biosynthetic pathways and that of many other genes which are not directly related to one-carbon metabolism. This may be due to prolonged culturing with glycine in the medium resulting in altered expression of genes mediated by one or more secondary factors. These may reflect an adaptive response rather than a direct consequence of glycine induction.
On the basis of the above data, a model for the mechanisms regulating glycine response is presented.
111 Publications
Hong, S-P., Piper, M.D. and Dawes, l.W. (1999) Control of expression of one-carbon metabolism genes of Saccharomyces cerevisiae is mediated by a tetrahydrofolate responsive protein binding to a glycine regulatory region including a core 5' CTTCTT -3' motif. Journal of Biological Chemistry 274, 10523-10532
Sinclair, D.A., Hong, S-P. and Dawes, l.W. (1996) Specific induction by glycine of the gene for the P-subunit of glycine decarboxylase from Saccharomyces cerevisiae .. Molecular Microbiology 19,611-623.
Hong, S-P., Piper, M.D. and Dawes, I.W. (1999) Identification of sequences responsible for nitrogen regulation of GCV2 encoding a subunit of glycine-catabolic enzyme complex in Saccharomyces cerevisiae. To be submitted
Hong, S-P., Winata, S., Piper, M.D. and Dawes, I.W. (1999) Genome-wide transcriptional analysis of one-carbon metabolism in Saccharomyces cerevisiae. To be submitted
IV Abbreviations
A adenosine BAS basal level transcription bp base pairs c cytosine c1 one-carbon ss/dsDNA single/double stranded deoxyribonucleic acid dNTP deoxynucleotide triphosphate Dmin YNB + glucose + NH4(S04)2 DHFR Dihydrofoalte reductase DNA deoxyribonucleic acid EDTA disodium ethy lenediaminetetraacetate EMSA electrophoretic mobility shift assay 5-CHO-THF AD-formyl tetrahydrofolate 10-CHO-THF NIO_formyl tetrahydrofolate G guanosme GCN general control non-repressed GDC glycine decarboxylase complex/glycine cleavage system GLYmin YNB + glucose+ glycine minimal medium GRR glycine regulatory region GSD glycine to serine conversion defective GTF general transcription factor Inr Initiator sequence kb kilobase pairs LPDH (dihydro )lipoamide dehydrogenase Min+gly/ser Dmin medium+ glycine/serine mRNA messenger RNA 5,10-CH-THF AD ,NlO-methenyl tetrahydrofolate 5,lO-CH2-THF AD ,Nl 0-methy lene tetrahydrofolate 5-CH_,-THF AD-methyl tetrahydrofolate NCR nitrogen catabolic repression N-source nitrogen source ONPG o-nitrophenyl-13-D-galactopyranoside OP-Cu I, 10-phenanthroline copper ORF open reading frame PEG polyethylene glycol PIC transcription preinitiation complex R punne RNA ribonucleic acid RNA pol II RNA polymerase II SDS sodium dodecyl-sulfate (c/m)SHMT (cytoplasmic/mitochondrial) serine hydroxymethyl transferase T thymidine THF Tetrahydrofolate UAS upstream activation X-gal 5-bromo-4-chloro-3-indoyl-B-D-galactopyranoside y pyrimidine YEPD/G yeast extract+ peptone+ glucose/glycerol medium YT yeast extract+ tryptone + NaCl
v ACKNOWLEDGMENTS
First and most of all, I would like to thank my supervisor Prof. Ian Dawes for giving me the opportunity to carry out this Ph.D. study in his laboratory. I could not have completed this work without his support, guidance, patience and enthusiasm throughout the years.
I am very grateful to Mel, Geoff and Chris for their helpful ideas and assistance during my postgraduate study. I also thank all the present and past members of labs 20 1 and 202; Hazel, Hal, Lisa, Machello (Claudio), John, Ji-Chul, Rachel, Vince, Nesrin,
Priyanka, Jacinta, Aner, Nazif, Tamara, Shahul, Steph, Jennifer, Gab, Sandra, Alexia, and Fiona for creating an enjoyable and pleasant working environment. I have been very lucky to work with you all.
My sincere and special thanks to Matt Piper, Maggie Evans, and David Sinclair for their friendship, humour and advice. Without them, it would have been much more difficult to carry out this work.
I also wish to thank to all those in the School of Biochemistry and Molecular
Genetics, for their assistance and help with use of equipment whenever needed. My thanks are extended to all those who provided me with strains or DNA for this research.
Finally, I can never thank my father and mother enough, for supporting me in numerous ways during my Ph.D. study.
My very special thanks to my beloved wife, Jung-In, for her patience, strength and smile- you were always there for me ...
Vl Introduction
Chapter 1: INTRODUCTION
1.1 Overview.
Modulation of gene expression at the level of transcription is a major regulatory strategy for eukaryotic cells to control their responses to intra- and extracellular stimuli. The level of transcription is mainly regulated by sequence-specific transcription factors (activators and repressors) binding to promoter elements via DNA-binding motifs to modulate transcription through direct or indirect (via coactivators) communication with the general transcription machinery.
The regulation of metabolic pathways is required for homeostasis and effective use of cellular components. In particular, the expression of genes for utilization of certain nutrients is tightly regulated to provide concerted gene expression to assist in an economic adaptation to the changing environment. There have been numerous molecular biological studies on the mechanisms of regulation of metabolic pathways in yeast, however, certain topics have received less attention than others, although they are no less important to the cell. One carbon metabolism which is crucial for the synthesis of nucleic acid, proteins, amino acids and vitamins is one that has been paid less attention, especially in eukaryotic organism.
This thesis is mainly concerned with an investigation of the transcriptional regulation of one-carbon metabolism, particularly that of the glycine decarboxylase multienzyme complex (GDC) which plays an important role as an entry point of glycine into one-carbon metabolism and in balancing between glycine and one-carbon units in the cell. This has also given an insight into the mechanisms by which the cell might control gene expression between different sub-metabolic pathways within the one-carbon metabolism.
In this study the budding yeast Saccharomyces cerevisiae has been used as a model system. Although many metabolic pathways are essentially identical in yeast and mammals,
1 Introduction
the breadth of molecular genetics available inS. cerevisiae offers powerful advantages over other systems.
1.2 One-carbon metabolism
Folate-mediated one-carbon metabolism plays an essential role in several major cellular processes including: nucleic acid biosynthesis (thymidylate and purines); the initiation of protein synthesis in bacteria and eukaryotic organelles (N-formylmethionyl-tRNAfmet); amino acid biosynthesis and interconversions (methionine, serine, glycine, and histidine); and choline and pantothenic acid metabolism.
1.2.1 Tetrahydrofolate, a carrier molecule of one-carbon metabolism.
The diversity of pathways of one-carbon metabolism is dependent upon the ability of the coenzyme, tetrahydrofolate (THF) which exhibits the most structural diversity of all the coenzymes. It can carry activated one-carbon units at either N-5 or N-10 positions of the pteridine ring and p-aminobenzoate groups, or by bridging these nitrogen atoms in different oxidation states from methanol (5-CH3-THF) through formaldehyde (S,lO-CH2-THF) to formate (5,10-CH-THF, 10-CHO-THF, and 5-CHO-THF) and cells can readily interconvert these forms (Figure. 1.1 ).
Animals lack the first three steps of THF synthesis, and hence folate supply in these organisms depends on feeding. In contrast, plants and microorganisms including
Saccharomyces cerevisiae are able to synthesize THF de novo. This pathway requires the sequential operation of five enzymes: dihydropterin pyrophosphokinase (HPPK), dihydropteroate synthase (DHPS), dihydrofolate synthetase (DHFS), dihydrofolate reductase
(DHFR) and folylpolyglutamate synthetase (FPGS). These enzymes are involved in the generation of pteridine precursors from GTP, the formation and reduction of DHF, and finally the glutamyl conjugation of THF. In pea leaves, the synthesis of 7,8-dihydropteroate by a single copy gene product has only been detected in mitochondria, indicating that mitochondria may be the unique site for its synthesis (Rebeille et al., 1997).
2 Introduction
(N-1) COOH
NH-tH I CH2 ~H2 COOH o=t-t-NH-tH I CH2 I CH2 I O=C-OH
R1 R2 Compound Abbreviation -H -H tetrahydrofolate THF
-CH3 -H 5-methy_ltetrahydrofolate 5-CH,-THF -CHO -H 5-formyltetrahydrofolate 5-CHO-THF -H -CHO 10-formyltetrahydrofolate 10-CHO-THF =CH- 5,1 0-methenyltetrahydrofolate 5,10-CH-THF -CH2- 5, l 0-methylenetetrahydrofolate 5,10-CH2-THF
Figure 1,1 Tetrahydrofolate (THF) with chemical structures and abbreviations, Chemical structure of THF is shown in the diagram. Single-carbon units can be carried at the different oxidation levels on THF on N-5 (Rl) or N-1 0 (R2), or bridged between N-5 and N-10. The table shows the compound names and abbreviations depending on the substitution at Rl and R2 ofTHF.
Clinical importance of one-carbon metabolism and THF inhibitors.
Folate deficiency in humans leads to a megaloblastic anemia which is caused by impairment of cell division and results in abnormally large red blood cell precursors with enlarged nuclei (Chanarin, 1990). It has been known that periconceptional supplementation with low doses of folic acid reduces the incidence of neural tube birth defects (Czeizel and
Dudas, 1992). One of the diseases that is caused by defects in one-carbon metabolism is hyperhomocysteinemia which results in elevation of both cysteine and homocysteine levels in the blood (homocystinemia) and in the urine (Finkelstein, 1990). Homocystinemia is associated with an increased risk of vascular disease and atherosclerosis (Wagner, 1995).
3 Introduction
Because of the role of the folate coenzymes in the synthesis of DNA precursors, folate antagonists have found widespread clinical use as anti proliferative and antimicrobial agents (Genther and Smith, 1977; Wooden et al., 1997). The extensive use of antifolates in cancer chemotherapy has stimulated considerable interest in the nature of folate and THF binding sites. Although the folate-binding sites of one enzyme in a variety of organisms seem to share functional and structural properties, enzymes with different activities have functionally distinct binding sites that probably do not have similar structures. Each folate dependent enzymes might have its own specific folate-binding site or the binding sites might rely on secondary or tertiary structure (Kirksey and Appling, 1996; Rebeille et al., 1994;
Takeishi eta/., 1985).
The chemotherapeutic value of antifolates including methotrexate (MTX) reside in their ability to perturb cytosolic folate-dependent one-carbon metabolism by competitively inhibiting the one-carbon pool enzyme DHFR. MIX has been used for treatment of cancer and a wide variety of non-neoplastic diseases such as rheumatoid arthritis, psoriasis, polymyositis, and asthma (Morgan and Baggot, 1995). MTX and other antifolates are thought to be cytotoxic because they suppress DNA synthesis and/or repair as a result of both pyrimidine and purine depletion (Schornagel and McVie, 1983; Taylor et al., 1982).
Induction of apoptosis has been shown to occur following culture of tumour cells with antifolates, which may be another role in the antitumour activity of these drugs (Dive et al.,
1992).
The role of polyglutamate tails of THF derivatives.
Previous studies have shown that the physiologically active forms of the THF derivatives are predominantly polyglutamates, rather than the monoglutamates (McGuire and
Coward, 1984) and the particular polyglutamates present in cells are characteristic of different species or culture conditions. For example, hexaglutamates predominate in S. cerevisiae and
N. crassa (Atkinson et al., 1995; Cossins, 1984), tetra- and penta-glutamates in peas
(Besson et al., 1993; !meson et al., 1990), and triglutamates in Clostridium species
4 Introduction
(Curthoys et al., 1972). There is also a different subcellular distribution of polyglutamyl
THF derivatives between the cytosol and mitochondria (Carl and Smith, 1995; Lin et al.,
1993), indicating that mitochondrial folate accumulation and metabolism is dependent on mitochondrial FPGS activity (Lowe et al., 1993).
The conversion of folates to their polyglutamate derivatives is facilitated by folylpoly y-glutamate synthetase (FPGS) by which glutamyl residues are linked by amide bonds through the y-carboxyl group. Metabolic turnover of these anabolites appears to be modulated by y-glutamyl hydrolase (GGH) after their mediated entry into lysosomes
(Barrueco eta!., 1992). A study with hog liver FPGS showed that the enzyme exhibited different binding rates for different folates, with a progressive decrease in rate upon further extension of the glutamate chain (Cichowicz and Shane, 1987b). Many eukaryotic FPGS enzymes showed preference for THF as substrate; for example, THF and DHF are the most effective substrates for human cytosolic FPGS (Chen et al., 1996). A mutant lacking this enzyme in different cell lines is auxotrophic for glycine, purines, thyrnidylate and methionine and the intracellular folate levels were reduced due to an inability to synthesize folylpolyglutamates (McDonald et al., 1995; Taylor and Hanna, 1977).
The role of the poly glutamate side chain of the coenzyme in regulation of one-carbon metabolism appears to be in altering the propeities of individual enzymes or in controlling the flux through different pathways. Other roles for folylpolyglutamates have also been suggested including storage forms of folate, effects on other enzymes, enzyme stabilisation and as allosteric effectors (McGuire and Beltino, 1981 ).
The polyglutamate tail of THF derivatives yield more efficient substrates for interconversion of folate coenzymes and available data indicate that the polyglutamy 1 forms of the THF coenzymes present in the cell may function in regulating the activity of particular enzymes with which they interact (Schirch and Strong, 1989). In general, polyglutamyl substrates are characterised by the same or only slightly greater V max values than those of the monoglutamate derivatives, while the Km values are decreased when polyglutamyl substrates
5 Introduction
are used (McGuire and Coward, 1984 ). They may also result in channelling of the coenzyme between several enzymes including multifunctional enzymes (Mackenzie and Baugh, 1980;
Schirch and Strong, 1989). Folate substrates containing glutamyl residues allow preferential transfer of intermediates between enzymes or in the case of multifunctional proteins, between active sites. The long anionic poly glutamate chain could provide not only tighter binding but also serve as a type of anchor to allow the tetrahydropteroy l moiety to release and bind to the next active site (Mackenzie and Baugh, 1980; Paquin eta!., 1985).
In addition, they are responsible for the cellular retention of folates in eukaryotic cells and organelles (McBurney and Whitmore, 1974; Taylor and Hanna, 1977), and it has also been suggested that one-carbon t1ux through the multiple folate-dependent reactions may be regulated by varying the glutamate chain lengths of folates under different conditions of growth or nutritional requirement (Cossins, 1984), although other authors argued that these changes are a secondary effect due to primary regulation of one-carbon metabolism at individual enzymes (Foo and Shane, 1982; Taylor and Hanna, 1977).
1.2.2 Donors of one-carbon units in S. cerevisiae. Major one-carbon donors in yeast
One-carbon units are obtained from serine, glycine, and formate in THF-mediated reactions. The serine-glycine pathway can be considered as a highly branched pathway with the central physiological role of producing most of the one-carbon units needed by cell
(Figure 1.2).
Serine is the major source of one-carbon units for cytoplasmic folate interconversions in most organisms (Schirch, 1984). Its 3-carbon is transferred to THF in a reversible reaction catalysed by serine hydroxymethyltransferase (SHMT) to generate the one-carbon loaded THF derivative, 5,10-CH2-THF, which is used for thymidylate synthesis or which can be reduced to 5-CHJ-THF for methyl group biosynthesis. Alternatively, 5,10-CH2-THF can be oxidised to 10-CHO-THF by C1-THF synthase. In rapidly growing cells, the
6 (glycolysis) isocitrate 3-phosphoglycerate .J,!3 1 glyoxylate (gluconegenesis)
II SERJ .J,! 4 I 12 SER2 phosphatidyl ~ ______phosphatidyl ~ 1 1 glycine ~ threonine choline serine 1 1 ..... I I - dTMP DHF ______.,_ THF 1.serinc f -- -~ ; ~!. -- - ff~------T~e serine
~ I ~ T (thymidylate) 4 "- ff S-AdoMet - dUMP glycme f -Jt glycine .______...,.. j.. 8 5,10-CH,-THF I ~J \ ~ 5,10-CH,-THF methionine ~ 5-CHJ-THF NA D?+ l NAD' i THF..j,. I ~ CO t NADp+ r 2 \ ~ NAD+ NADP ~~ ~00~ ~ ~~ Homocysteine 5-CHO-THF f __ .. 5 lO-CH-THF ~ 5 10-CH-THF
) 10-CHO-THF ~ 10-CHO-THF histidine T£ ,_. ADP \ ADP l j TH F ~ATP ~ _ ATP THF. ff formate f------~-- t formate -
Figure 1.2 Pathway of folate-mediated one-carbon metabolism in yeast. The major enzyme reaction in the pathway are indicated: 1, gl ycine decarboxylase complex (encoded by GCVJ, GCV2, GCV3 and LPDJ); 2, mitochondrial serine hydroxymethyltransferase (SHMJ); 3, mitochondrial C1-THF synthase (MJSJ); 4, cytoplasmic serine hydroxymethyltransferase (SHM2) ; 5, cytoplasmic C1-THF synthase (AD£3) ; 6, NAD-dependent CH2-THF dehydrogenase (MfDJ); 7, dihydrofolate reductase (DFRJ); 8, 5,10-CH2-THF reductase (MEFJ3); 9, thyrnidylate synthase (TMPJ); 10, homocysteine methyltransferase (MET6); 11 , S-adenosylmethionine synthetase I (SAMJ); 12, threonine aldolase (GLYJ); 13, isocytrate lyase (JCLJ); 14, glyoxylate transaminase. Red types indicate the major one-carbon donors and blue types are for the major one-carbon metabolic products. Introduction
synthesis of purines is a critical function of folate-dependent one-carbon metabolism, requiring 2 moles of 10-CHO-THF per mole of purine ring.
Glycine can serve as a source of one-carbon units (Ogur et al., 1977). It is broken down by the mitochondrially located glycine decarboxylase (GDC; also called glycine cleavage system or glycine synthase), producing 5, lO-CH2-THF. The GDC serves an important role in balancing cellular requirements for glycine and one-carbon units. The ability of a high level of glycine to substitute for the serine requirement in a serl strain (a mutant which can not synthesize serine through the glycolytic pathway) was demonstrated in
S. cerevisiae (Ulane and Ogur, 1972). Under this condition, the GDC plays an important role by cleaving some of the glycine into C02 and 5,10-CH2-THF, thus providing one carbon units to the cell and the S,lO-CH2-THF is also subsequently used for serine synthesis by SHMT by reaction with another glycine molecule (Ogur et al., 1977; Pasternack et al.,
1992).
Formate can also serve as a one-carbon donor, entering the active pool at the level of
10-CHO-THF by the reaction catalysed by the 10-CHO-THF synthetase activity of C 1-THF synthase (McKenzie and Jones, 1977). Indeed, formate can satisfy all its one-carbon requirements via this pathway in a serl strain (Appling and Rabinowitz, 1985; Barlowe and
Appling, 1990; McKenzie and Jones, 1977).
Under normal growth conditions, it is likely that cells utilise serine, glycine, formate and perhaps other one-carbon donors simultaneously, but to different extents and in different compartments. It was shown that the contribution from each one-carbon donor is different for each of the one-carbon pools (Pasternack eta/., 1996). The state of equilibrium between one-carbon pools and the donors in a growing cell depends on the source of the one-carbon units.
There are other potential sources of one-carbon units, such as histidine (carbon 2 of the imidazole ring) or tryptophan (carbon 2 of the indole ring) (Appling, 1991; Schalinske and Steele, 1996). In human, choline may be a significant source of one carbon units both in
7 Introduction
the cytosol and mitochondria, and dimethylglycine dehydrogenase, sarcosine dehydrogenase reactions as well as the glycine cleavage system also catalyse the supply of one carbon units in mitochondria (Park and Garrow, 1999).
Source of one-carbon donors in S. cerevisiae
There are three known pathways for serine biosynthesis inS. cerevisiae. During growth on fermentable substrates, yeast cells use 3-phosphoglycerate from glycolysis to produce the majority of serine. The first enzyme of this pathway, 3-phosphoglycerate dehydrogenase is subject to feedback inhibition by serine (Melcher and Entian, 1992). The serl and ser2 mutant strains are blocked in the second (3-phosphoserine transaminase) and the third enzyme (3-phosphoserine phosphatase) respectively for serine synthesis and when grown on glucose, the majority of serine must be synthesised from glycine via GDC (Ulane and Ogur, 1972). Northern analysis showed serine represses transcription of SERI three fold (Melcher et al., 1995). Secondly, during growth on non-fermentable carbon sources, the major source of serine is the glucose-repressible gluconeogenic pathway, in which glyoxylate from the anaplerotic glyoxylate cycle is converted to glycine and then to serine by SHMT
(Ulane and Ogur, 1972). Thus, strains which were serine-glycine dependent in glucose media became capable of serine-glycine independent growth on acetate media, which was termed conditional auxotrophy. A third minor pathway may proceed via a vanadate dependant serine sulfhydrase which is possibly encoded by SERIO, converting cysteine to serine (Meisch and Kappesser, 1987; Melcher and Entian, 1992).
The first two pathways in serine biosynthesis described above are also important for glycine biosynthesis. Serine synthesised in cells grown on a fermentable carbon source via the glycolytic pathway is converted to glycine by SHMT which cleaves serine into glycine and S,lO-CH2-THF. Recently, a third pathway of glycine synthesis was found. A threonine aldolase encoded by the GLYI gene was found by using a knock-out mutant auxotrophic for glycine and by searching the yeast genome for sequence homology to threonine aldolase from other organisms (Liu et al., 1997; Monschau eta/., 1997). GLYI is thought to be the major
8 Introduction
source of glycine in yeast since disruption of GLY1 alone led to a strongly reduced growth
rate in the absence of glycine whereas disruption of both SHM genes (encoding
mitochondrial and cytoplasmic SHMT) did not, even when there was no limitation in one
carbon pools (McNeil et al., 1994).
1.2.3 One-carbon flux in S. cerevisiae.
In eukaryotes, the cytoplasmic and mitochondrial compartments each possess a parallel array of enzymes that utilise one-carbon units in different oxidation states; the
reactions of many of these are reversible (Figure 1.2). Since THF derivatives do not cross
the mitochondrial membrane to any significant extent (Cybulsky and Fisher, 1981; Horne et
a/., 1989), transport of one-carbon units between compartments relies on one-carbon donors
such as serine, glycine or formate. The amino acids glycine and serine are rapidly
transported across the mitochondrial membrane (Cybulsky and Fisher, 1976).
The derivatives of THF that predominate in the cytoplasm and mitochondria differ
(Horne et al., 1989), as does the distribution of folate-dependent enzymes between the two organelles. For example, in S. cerevisiae, the total activity of mitochondrial SHMT
(mSHMT) is 5% of the total cellular (cytoplasmic and mitochondrial) SHMT activity,
although mSHMT has an approximate two-fold higher specific activity (Kastanos et al.,
1997). The cytoplasm and mitochondria each contain enzymes with identical activities, and each set of enzymes of one-carbon metabolism is directed toward establishing a compartment-specific distribution ofTHF derivatives.
In vivo NMR studies provide a useful system to study one-carbon flux due to its ability to distinguish small changes in intracellular concentration of !3C-label at specific carbon atoms in metabolites. Together with the genetic studies of one-carbon metabolism, it provide invaluable information on one-carbon flux in the cell. Whereas the C1 and C4 positions of choline and C2 and Cs positions of purines are derived from the one-carbon pools, Cs of purine and C2 of choline come from the direct incorporation of glycine and
9 llllroduction
serine respectively, hence the extent to which these positions are labelled reflects the extent to
which the local one-carbon pools are labelled.
One-carbon flux through SHMT isozymes
Growth and !3C-NMR experiments indicate that the mSHMT and cSHMT function in
different directions, depending on the nutritional conditions of the cell (Kastanos et al.,
1997). In a 13C-NMR study with [2-13C] glycine, yeast grown on serine as the primary
one-carbon source showed the cytoplasmic SHMT (cSHMT encoded by SHM2) was the
main provider of glycine and one-carbon units from the breakdown of serine. Although the
mitochondrial SHMT (mSHMT encoded by SHMl) also catabolizes serine, its contribution
is not as important under these conditions (Kastanos et al., 1997). However, when serine is
limiting, i.e. in cells grown on glycine, the mitochondrial SHMT was the predominant
isozyme catalysing the synthesis of serine from glycine and one-carbon units (McNeil et al.,
1996). When both serine and glycine were present, the mSHMT made a significant
contribution to the one-carbon pools, but not glycine, for purine synthesis (Kastanos et al.,
1997).
Cytoplasmic and mitochondrial one-carbon flux
When cells were grown in media with serine or glycine as a sole one-carbon source,
an shm2 mutant showed slightly slower growth (about 1.5 times) than the wild type, but an shm2, misl strain did not grow at all. Both strains were restored to wild type growth when
supplemented with formate, inferring the role of the mitochondrial pathway in generating mitochondrial formate for use in the cytoplasm (McNeil et al., 1996). 13C NMR studies
showed that mitochondrialS,lO-CH2-THF is oxidised to formate and then transported to the cytoplasm. 13C-NMR analyses with various yeast mutants showed that at least 25% of one carbon units utilised for cytoplasmic purine synthesis are derived from mitochondrial formate regardless of serine availability (Pasternack et al., 1994a). These analyses also showed the incorporation of C02, 10-CHO- THF and glycine into purines in yeast, and that unlabelled
10 Introduction
formate could compete out the labelling of the C2 and Cs of purine from the C2 of glycine
(Kozluk and Spencer, 1987).
In yeast, the GDC reaction is reversible and cells expressing mitochondrial C 1-THF
synthase (encoded by MIS!) activity incorporate 13C-formate into [2-l3C] glycine.
Furthermore, cells supplied with [3-l3C] serine produced [2-13C] glycine and [2,3-l3C]
serine. These data imply that the mitochondrial pool of S,10-CH2-THF arising from formate
or serine is in equilibrium with the C-2 of glycine and the C-3 of serine (Pasternack et al.,
1994a; Pasternack eta/., 1992).
In a mutant lacking both cytoplasmic and mitochondrial C 1-THF synthases, there was no assimilation of label into cellular one-carbon metabolic products when cells were exposed to [l4C] formate. This result confirms that one or both of the C1-THF synthases are required for formate utilisation (Pasternack et al., 1992). Metabolism of [13C] formate to [2-l3C] glycine and [3- Uq serine in a strain lacking the cytoplasmic C 1-THF synthase shows that formate does indeed enter and is metabolised in mitochondria in vivo (Pasternack et al.,
1992). This result also provides evidence for the synthesis of glycine via reversal of GDC using 5,10-[l3C]CH2-THF, C02, NH4+ as substrates, in vivo. However, mitochondrial
C 1-THF synthase represents only about 6 to 10% of the total C 1-THF synthase activity in a wild type strain (Pasternack et al., 1994b; Shannon and Rabinowitz, 1986), and since formate cannot satisfy the one-carbon requirement of a serf, ade3 strain, flux through this enzyme is rate-limiting in providing sufficient serine in this strain (Barlowe and Appling,
1990; Pasternack eta!., 1992). Furthermore, in a serl, misl strain, growth was not affected on formate (Shannon and Rabinowits, 1988). Therefore, when formate is the one-carbon donor, assimilation is primarily cytoplasmic. Also, analysis of serl, shml and serl, shm2 strains for suppression of serine auxotrophy by formate in a serl strain revealed that cSHMT but not mSHMT is essential for this effect (Table 1.1). Therefore, the prevailing flux of mitochondrial folate interconversion is from S,lO-CH2-THF to formate (McNeil eta!.,
1996).
11 Introduction
1.2.4 Compartmentation of one-carbon metabolites: a proposed model
Surprisingly, the purification of enzymes of one-carbon metabolism and determination of the size of the one-carbon pools showed that the concentration of the enzymes actually exceeds the concentration of the one-carbon folate pools. This raised the question of whether the rate of one-carbon metabolism is actually controlled by diffusion of substrates and products from these enzymes or via some type of transfer mechanism.
Substrate channelling
Substrate channelling is the local flux of metabolic intermediates through sequential enzyme reactions and provides a dynamic microcompartmentation (Spivey and Merz, 1989).
This involves multifunctional enzymes or multienzyme complexes in which the metabolic intermediate is passed directly to the next enzyme or active site without dissociating from the complex.
Substrate channelling offers a number of potential kinetic and regulatory advantages including high metabolic rates with low subslrate concentrations due to the independency of bulk phase substrate concentrations, a negligible lag-time in coupled reactions and isolation from competing reactions. In addition, unstable intermediates may be protected and the pathway can be regulated by changes in enzyme associations as metabolic conditions change
(Spivey and Merz, 1989). Because of THF derivatives' lability and their relative low cellular concentrations together with existence of multienzyme complexes and multifunctional enzymes in one-carbon metabolism, it was suggested that one-carbon unit transfer is mediated by channelling (Appling, 1991).
Implication of microcompartmentation in S. cerevisiae
The compartmentation of THF has been suggested in studies with the C1-THF synthase I SHMT coupled reaction. There was a huge discrepancy between the expected local THF concentration needed to obtain the experimentally observed flux through this coupled reaction and the known intracellular reduced folate concentration in S. cerevisiae,
12 Introduction
suggesting not all the THF is available (compartmented) to this coupled reaction (Pasternack
eta!., 1994b ).
In a wild type strain grown with [2-13C] glycine as a sole one-carbon source, as
unlabelled formate was added, relative enrichment for all three oxidation states of one-carbon
pools decreased. Exogenous 5-CHO-THF can serve as a major one carbon donor when the
cellular pools of THF are depleted by antifolates. In this case, 5-CHO-THF is mostly
metabolised to a I 0-CHO-THF pool available for purine synthesis, thus acting to reverse the
effects of antifolate and very little of the 10-CHO-THF is converted to more reduced one
carbon pools. These experiments provide indirect evidence for the compartmentation of the
one-carbon pools. Although formate and 5-CHO-THF enter the one-carbon pools via 10-
CHO-THF, the final distribution differs, implying multiple pools of 10-CHO-THF exist
(Pasternack eta!., 1996).
The wild-type strain (SERJ, ADE3) synthesises three labelled species of serine from
[ 13C] formate: [3-13C] serine, [2-UC] serine and [2,3-13C] serine. [2,3-13C] serine must be produced via SHMT from [2-13C] glycine and 5,10-[l3C]CH2-THF. On the other hand,
strain lacking cytoplasmic C1-THF synthase, while synthesising [2-!3C] glycine, only produces [3-13C] serine. The lack of [2,3-13C] serine in this strain indicates that only unlabelled glycine was used in the mSHMT reaction, despite the presence of [2-13C] glycine produced in the mitochondria. This result indicates that there are two pools of glycine, only one of which is available for mitochondrial serine synthesis (Pasternack eta/., 1992).
Another 13C-NMR analysis of one-carbon metabolism showed that there are two pools of glycine and serine in the mitochondria. When cells were grown with [2-13C] glycine as a sole one-carbon donor, it was shown that there are two pools of serine, one pool is directed to choline synthesis and the second pool accumulates as serine. Likewise, incorporation of glycine into serine versus choline indicated the presence of two glycine pools in the mitochondria, and for each pool of glycine, the 5, I O-CH2-THF produced in the
GDC reaction may be channelled directly to SHMT without mixing with 5, 10-CH2-THF
13 Introduction
produced in the other glycine pool. Tight association of the GDC and SHMT could explain this highly interesting situation (Pasternack et al., l994a).
1.2.5 Genetic studies of one-carbon metabolism in S. cerevisiae.
Analyses of the nutritional requirements and metabolic defects in mutant strains affected in the tetrahydrofolate interconverting enzymes allows definition of the routes by which one-carbon units move between the different one-carbon pools within the cell. Table
1.1 summarises such analyses inS. cerevisiae.
Utilisation of glycine by mitochondria
As long as the cytoplasmic set of enzymes involved in one-carbon metabolism remains intact, the products of mitochondrial folate interconversion are not essential for folate-dependent anabolic pathways of the cytosol (Table 1.1 ).
In minimal medium with or without serine, the shml, shm2 double mutant shows a moderate growth defect which is partially relieved by addition of glycine. However, shml, shm2, mis 1 or shm2, shml, gcv 1 triple mutants did not grow at all even with the glycine supplement, suggesting that glycine is utilised by the mitochondrial pathway (via GDC and mitochondrial C1-THF synthase) (McNeil et al., 1996). In addition, since shm2, misl has a longer generation time than the shm2, shml strain, it is believed that the MISJ product is responsible for a major portion of mitochondrial formate synthesis (McNeil et al., 1996).
Growth was restored in a serl strain, but not as much as in serine even at high level of glycine as sole one-carbon source because serine and one-carbon units remain limiting in the cytoplasm although mSHMT and GDC can satisfy the mitochondrial requirement.
Formate could fully restore the growth of this strain. However, formate only partially rescued the growth of the strain lacking mSHMT (serl, shml), indicating that cSHMT is not able to fully satisfy the cell's serine needs in the absence of mSHMT. If cells were grown in serine, mutants lacking cSHMT showed limited growth regardless of the rate of serine cleavage by mSHMT (Kastanos et al., 1997).
14 Table 1.1. Phenotypes of one-carbon metabolic mutants
Mutations Phenotype References shmt No effect on qrowth. McNeil et at. (1996) shm2 Weak formate auxotrophy. Neither serine nor gly_cine suj)portJlrowth. McNeil et at. 0996) gcvt No effect on growth. McNeil eta/. (1996) Unable to qrow on qlycine as a sole nitrogen source lGL Ymint McNeil et at. .b 997) mist No effect ongrowth. McNeil et at. (1996) sert High level of exogenous glycine or formate fulfill the serine requirement, serine restores Ulane and Ogur (1972) wild type growth. Did not grow in Dmin + glyoxylate or adenine. Grows slowly without McNeil eta/. (1996) any supplement in nonfermentable carbon source (eq. acetate) Kastanos et at. (1997) otvt Serine or qlycine restored qrowth to wild-type level, but formate or adenine did not. McNeil et a/.11996) dfrt Mutant in dihydrofolate reductase (DHFR). Auxotrophic requirement for dTMP, Huang et at. (1992) adenine, histidine and methionine. Petite phenotype. shmt, shm2 Moderate growth defective in minimal media (Dmin) and Dmin + ser. Glycine can partially McNeil eta/. (1996) restore qrowth. Formate restored the wild-type qrowth. shm2,gcvt Weak formate auxotropt1y (+_glycine decreases growth rate). McNeil et a/.11996) shmt, shm2, ocvt Neither of glycine or serine restored growth but formate did. McNeil eta/. (1996) mist, sert No distinguishable phenotype from sert strain (even on nonfermentable carbon Shannon and Rabinowits source). (1988) mist, shm2 (or) Complete formate auxotrophy, neither glycine or serine restored growth but formate or McNeil et at. (1996) mist, shmt, shm2 adenine did. mist, shmt No effect on qrowth McNeil et at. (1996) sert, shmt Formate partially fulfills the serine requirement (serine restores WT growth). McNeil eta/. (1996) High level of qlycine did not fulfill the serine requirement. Kastanos eta/. (1997) ser1, shm2 Glycine fulfills the serine requirement. Formate did not support the growth. McNeil et at. (1996) Serine+ formate or hi(lh level of glycine+ formate restores the wild-type growth Kastanos et at. (19971 sert, shmt, shm2 Formate + serine required to achieve growth rate of wild-type growth (serine alone was McNeil eta/. (1996) not enough), no qrowth on high level of glycine +formate. Kastanos et at. (1997)_ ser1, _qcv1 Serine auxotrophy (was not fulfilled by exoqenous qlycine). McNeil et at. (1997) g/yt, shmt Partial glycine auxotroph, serine or formate did not fulfill glycine requirement. McNeil et at. (1994) McNeil et at. h 996) glyt, shm2 Glycine auxotroph, serine or formate did not fulfill glycine requirement. McNeil et at. (1994) McNeil et at. /1996) g/yt, shm t, shm2 Complete glycine auxotroph (relieved partially when cells were grown in non McNeil eta/. (1996) fermentable carbon source eq. ethanol). Monschau et at. (1997) Mutations Phenotype References ade3, ser1 Adenine auxotroph, not overcome by glycine+ formate or glycine+ serine. Barlowe and Appling (1990) Did not orow in adenine+ qly_cine but_grew in glycine+ adenine+ serine. Pasternack et at. (1994al ade3, ser1, mis 1 Adenine auxotroph, not overcome by glycine +formate or glycine + serine. Barlowe and Appling (1990) Grow in adenine + qlycine or qlycine + adenine+ serine Pasternack eta/. (1994a) ser1, S Grow in the absence of adenine, very slow growth in glycine + formate, only restored Barlowe and Appling (1990) growth by serine addition (did not grow in adenine + glycine but grow in glycine + West eta/. (1996) adenine+ serine). Pasternack et at. '(1994a) ser1, N Grow similarly to ser1 in all tested conditions. West eta/. (1996) sert, 0 Grow in the absence of adenine, slow growth in glycine + formate, slower growth in Barlowe and Appling (1990) ' glycine+ formate than sert, N (N does not contribute to reductive reaction.) ser1, N, 0 Show growth in glycine +formate, grow in adenine+ glycine. West eta/. (1996) Grow slower in Dmin +serine, compared to sert, Nor sert, 0 (depends on mitochondrial Pasternack eta/. (1994a) pathway which is limiting for growth; grow normally by the additional supplement of adenine). ser1, N, 0, S Did not grow in Dmin +serine, or glycine+ formate and adenine auxotroph. West eta/. (1996) Grow normally in serine + adenine. sert, S, 0 (or) Grow in the absence of adenine, very slow growth in glycine+ formate. Barlowe and Appling (1990) sert, S, N Grow normally with serine addition. West eta/. (199S) sert, S, 0, C Grow in the absence of adenine, but not grow in glycine +formate or adenine + glycine. Barlowe and Appling (1990) Doubling time was increased only 2 times in Dmin + serine compared to sert, S, 0 strain West eta/. (1996) Pasternack eta/. ·(1994a) sert, S, 0, C, N No growth on glycine +formate, did not restore the growth by serine. West eta/. (1996) Adenine alone partially, adenine + serine fullv restores orowth. Pasternack eta/. ·(1994a) tup dTMP permeable_ (wild-type strains usually impermeable). Little and Haynes_i1979l ade3, shm t, tup Adenine auxotrophy. McNeil eta/. (1996) ade3, shm2, tup_ Methionine and adenine auxotrophy, prototrophy for dTMP. McNeil et at. 1996 ade3, shmt, shm2, tup Methionine, dTMP and adenine auxotrophy. McNeil eta/. 1996 ade3, shm1, mist, tup Adenine auxotrophy. McNeil et at. 1996 ade3, shm2, mis 1, tUfJ_ Methionine and adenine auxotrophy, prototrophy for dTMP. McNeil eta/. (1996) ade3, shmt, shm2, Methionine, dTMP and adenine auxotrophy. McNeil eta/. (1996) mist, tup
S, synthetase mutant of cytoplasmic C1-THF synthase; D, dehydrogenase mutant of cytoplasmic C1-THF synthase; C, cyclohydrolase mutant of cytoplasmic C] THF synthase; N, disruption of cytoplasmic NAD-dependant dehydrogenase. The name of the enzyme encoded by each gene is shown in Figure 1.2. Introduction
Whereas glycine could not increase the growth rate of a shm2 mutant through its
conversion to formate, glycine did satisfy the serine requirement of serl yeast, suggesting
that 5,lO-CH2-THF derived from glycine cleavage may be directed towards the synthesis of
serine (via mSHMT) rather than formate. This is supported by the observation that glycine
did not substitute for the serine requirement in either serl, gcvl or serl, shml strain, but
could replace serine in a serl, misl background. McNeil eta/. (1996) proposed that the
GDC and mSHMT channel glycine through sequential reactions to yield serine which can be
utilised in the cytoplasm. Interestingly, shml, shm2 yeast possess a shorter doubling time
than the shm2 single mutant in the presence of glycine. The authors suggested that in an shml null mutant, substrate channelling of glycine is uncoupled and the 5,10-CH2-THF is
now available to C1-THF synthase and the resultant formate is transported to the cytoplasm.
In pea leaf mitochondria, the major function of mSHMT is to recycle 5,lO-CH2-THF produced by the GDC, to THF to allow the continuous operation of the glycine-oxidation pathway. However, the rate constant of the reaction converting 5, !O-CH2-THF to THF was
15 times lower than the rate constant for the reverse reaction, therefore the mSHMT reaction must be permanently pushed out of equilibrium towards the production of serine and THF to
allow the whole process to take place (Besson eta/., 1993).
Role of the C1-THF synthase in one-carbon metabolism
It has been suggested that the C 1-THF synthase has both catalytic and noncatalytic
functions in de novo purine synthesis. The noncatalytic function is via the formation of a purine synthesising multienzyme complex or "metabolon", in which C1-THF synthase is required as a structural component. This model predicts that in the absence of the C 1-THF synthase protein, regardless of 10-CHO-THF availability, an active complex is unable to form, resulting in purine auxotrophy (Barlowe and Appling, 1990).
The catalytic function of the NADP-dependent 5,1 O-CH2- THF dehydrogenase activity of C1-THF synthase can be replaced by the monofunctional NAD-dependent dehydrogenase encoded by the MTDJ gene (West et al., 1993). Growth studies (table 1.1;
15 Introduction
compare serf. D with either serf or serf, N) showed that these dehydrogenases are
interchangeable when flow of one-carbon units is in the oxidative direction but not in the
reductive direction (West et ul .. 1996). The existence of two dehydrogenases would allow
shifting of the interconversion of one-carbon units toward either the more oxidised form for
de novo purine synthesis or the more reduced form for methyl group generation or
thymidylate synthesis as conditions change. When both dehydrogenases are inactive. the cell
must rely on the mitochondrial pathway for one-carbon metabolism which is limiting for
growth (Table 1.1; serl, N. D).
One thing to note is that at physiological pH, the equilibrium between 5.10-CH-THF
and 10-CHO-THF lies far towards 10-CHO-THF (Kay et al.. 1960), and the nonenzymatic
hydrolysis of 5,10-CH-THF to 10-CHO-THF is quite rapid (Robinson, 1971). These facts
can explain the decrease in growth rate of the serl. S. D. C strain compared to serl. S. Din
minimal medium with serine (Table 1.1 ).
McKenzie and Jones (1977) showed that all mutants that were defective in
dehydrogenase activity also lacked cyclohydrolase activity. Other studies based on use of
site-specific mutations, immuno-titration and chemical treatments further provided evidence
for an overlapping cyclohydrolase-dehydrogenase site that is independent of the synthetase
active site (Appling and Rabinowitz, 1985; Barlowe et al., 1989). Similar results were
obtained from the human and porcine enzymes (Pelletier and MacKenzie, 1994; Smith and
MacKenzie, 1985).
There is an implication of a more global connection between cytosolic synthetase
function and utilisation of mitochondrial one-carbon units. In table 1.1, growth of various
serl mutants with different combinations of mutations in the C 1-THF synthase is shown.
Tests for their ability to substitute exogenous glycine for serine (independent of adenine
auxotrophy) revealed that strains lacking cytoplasmic synthetase activity of C 1-THF synthase but containing the mitochondrial C 1-THF synthase were unable to grow on minimal medium plus glycine and adenine (compare ade3, serl, misl strain with serl. ade3 or serl, S or serl,
16 Introduction
S, D, C), which indicates that the mitochondrial oxidation of CHz-THF via mitochondrial
C I-THF synthase, along with the inability to activate formate in the cytosol, prevents growth suggesting a link between two enzymatic events (Pasternack et al., 1994a).
1.2.6 Regulation of one-carbon metabolism
The regulation of C I-THF synthase appears to require the simultaneous presence and absence of several one-carbon products. Excess adenine, methionine, histidine and pantothenate act in concert repress the enzyme (2-3 fold difference in enzyme concentration and mRNA level) indicating that the cell can determine the cellular levels of each these four nutrients perhaps by monitoring a common metabolite (Appling and Rabinowitz, 1985). The regulation of C I-THF synthase suggests that the levels of the various coenzyme forms of
THF are very tightly controlled. This is also supported by the observation that drug-induced folate starvation causes significant derepression of the enzyme (Appling and Rabinowitz,
1985).
One of the one-carbon metabolic intermediates, 5, 10-CHz-THF is a principal source of one-carbon units and an important branch point in folate-dependent metabolism.
Depending on the needs of the cell, it can be utilised biosythetically for methionine and de novo dTMP synthesis or oxidised to form 10-CHO-THF for purine synthesis. It has been suggested that the cellular concentration of 5,10-CHz-THF may regulate the flux of the coenzyme between the pathways leading to nucleotide biosynthesis and methionine regeneration. According to this prediction, low concentrations of 5, 10-CHz-THF will limit t1ux of one-carbon units into the purine biosynthetic pathway (Green et al., 1988).
Folate concentrations rapidly increased when cells were transferred from a methionine-containing medium to one lacking methionine and also, in the presence of methionine, inhibition of 5, 10-CHz-THF reductase was observed, indicating that the folate pool size was to some extent regulated during growth in the presence of exogenous L methionine (Lor and Cossins, 1972). The authors suggested that methionine directly or indirectly exerts a feedback inhibition on the enzymes required for synthesis of 5-CH3-THF.
17 Introduction
In other words, exogenous methionine decreases the flow of one-carbon units through the
methyl-folate pool by affecting the concentrations and activities of folate enzymes. The repression of S-CH3-THF homocysteine methyltransferase by methionine would provide a
further mechanism for regulation of methionine biosynthesis. Methionine would principally regulate one-carbon metabolism by controlling the reduction of 5, l0-CH2-THF. An experiment using l4C-formate showed that in the presence of methionine the flow of one carbon units was diverted from methyl-group biosynthesis to a more extensive use in the
synthesis of serine and the adenosyl-moiety of S-adenosylmethionine (Lor and Cossins,
1972).
1.3 Glycine decarboxylase multienzyme complex (GDC)
1.3.1 The role of GDC
The glycine decarboxylase multienzyme complex (GDC; EC 2.1.2.10) also called glycine synthase or glycine cleavage system is responsible for the catalytic conversion of glycine to yield carbon dioxide, ammonia, NADH and S,10-CH2-THF in a reversible reaction (Kikuchi, 1973; Koichi and Kikuchi, 1974). The GDC is composed of four different subunits known as the P-protein, H-protein, T -protein and L-protein (Figure 1.3).
The GDC was first described in cell-free extract of an anaerobic bacterium, Peptococcus glycinophilus (Sagers and Gunsalus, 1961) and has subsequently been found in the inner membrane of mitochondria of various animals (Richter et al., 1962; Yoshida and Kikuchi,
1972) and plants (Cossins and Sinha, 1966; McConnell, 1964) as well as within the cytosol of a number of bacteria (Jones and Bridgeland, 1966; Pitts and Crosbie, 1962).
Molecular defects in the GDC of humans leads to nonketotic hyperglycaemia (NKH) which is an autosomal recessive metabolic disorder in which there is a large accumulation of glycine in body fluids resulting in various neurological symptoms such as convulsive
18 Introduction
seizures, apnea, and mental retardation in young infants (Nyhan, 1989). It has been shown
that more than 80% of patients with NKH have defects in the P-protein (Tada, 1987).
In plants, the GDC is a key enzyme for the decarboxylation step of the
photorespiratory C-2 pathways, and is present in very large amounts (30-50% of total
soluble protein) in the mitochondrial matrix of illuminated leaves of pea (Bourguignon eta/.,
1988; Oliver eta!., 1990a). The concentration of the complex in plant tissue is controlled by
light. Upon illumination of etiolated leaves the enzyme activity increases about 10-fold
(Walker and Oliver, 1986), which is largely regulated at the transcriptional level
(Bourguignon eta!., 1993; Kim and Oliver, 1990). The GDC is stable in mitochondrial matrix of pea with an approximate subunit ratio of 2 P-protein dimers : 27 H-protein monomers: 9 T-protein monomers: 1 L-protein dimer (Oliver eta!., 1990a; Oliver et al.,
1990b).
C02
coo- l H-C-NH3+ I H P-Protein H-Protein H coo- Is PLP~ =N-~-H ip I 5, 10-CHrTHF I 'S H
L-Protein FAD• , NAD NADH + W
Figure 1.3 Reaction model for GDC. P-protein, which contains pyridoxal phosphate (PLP) as a cofactor, catalyzes the decarboxylation of glycine and transfers the remaining aminomethyl moiety to one of the sulfhydryl groups of the lipoyl prosthetic group of H-protein. The aminomcthyl moiety is cleaved by the action ofT-protein in the presence ofTHF, yielding ammonia and 5,10-CHz-THF. The dihydrolipoyl group of H-protein is reoxidised by L-protcin. Lip, lipoamide; THF, tetrahydrofolate.
19 Introduction
The P-protein of GDC
The P-protein is considered the true glycine decarboxylase. By itself it is almost inactive, but when the H-subunit is present, there is a change in the prosthetic group, pyridoxal phosphate, and is converted to the active enzyme (Hiraga and Kikuchi, 1980b ).
The a-amino group of glycine forms a Schiff-base with a pyridoxal phosphate coenzyme of the active site of the P-protein. The P-protein catalyses the exchange of the carboxyl carbon of glycine with C02 where upon the remaining methylamine moiety of glycine is transferred to one of the sulfhydryl groups of the lipoic acid prosthetic group of the H-protein (Hiraga and Kikuchi, 1980a). Genes encoding the P-protein of GDCs have been cloned from chicken and human (Kume et al., 1991), pea (Turner et al., 1992a), daisy (Kopriva and
Bauwe, 1994), yeast (Sinclair eta/., 1996) and E. coli (Okamura-Ikeda eta!., 1993).
Amino acid sequence analysis of the P-protein showed that it has structure characteristic of pyridoxal 5'-phosphate dependent amino acid decarboxylase, tryptophan synthase and serine hydroxymethyltransferase (Fujiwara et al., 1987). Many glycine residues and hydrophobic amino acids are found at the C-terminal side of the phosphopyridoxyl-site, and it has been suggested that this confers steric freedom facilitating the approach of the lypoyl moiety of the H-protein. Comparison of all known P-proteins reveals the presence of a highly conserved canonical (LIM)-X6 domain and this leucine zipper domains probably participate in P-protein dimerization (Bauwe et al., 1995). There are other reports of the involvement of leucine-zippers in protein-protein interactions for other than DNA-binding proteins (Konstantinov and Moller, 1994).
The H-protein of GDC
The pivotal enzyme which interacts with each of the other three proteins of the GDC is the lipoamide-containing H-protein. The lipoamide group in its oxidised form interacts with the glycine-loaded P-protein, the methylamine group of glycine is passed to the lipoyl prosthetic group of the H-protein. The lipoic acid is reduced during this transfer (Hiraga and
Kikuchi, 1980b). The amino acid sequence around the lipoate attachment site is highly
20 Introduction
conserved and predicted to form a B-hairpin loop. It has been suggested that such a structure might play a crucial role in the movement of the lipoic acid prosthetic group from one site of the GDC to another (Fujiwara et al., 1990; Pares et al., 1994).
When diluted, the GDC tends to dissociate into its component enzymes. In the dissociated state, the H-protein acts as a mobile cosubstrate that commutes between the other three enzymes and shows typical substrate kinetics. On the other hand, when the complex is reformed, the H-protein no longer acts as a substrate but as an integrated part of the enzyme complex (Oliver et al., 1990b).
The H-protein encoding genes have been identified in pea, chicken and yeast
(Macherel et al., 1992; Nagarajan and Storms, 1997; Yamamoto et al., 1991). In human, only one H-protein gene has been identified, but there are additional genes similar to the H protein eDNA (Koyata and Higara, 1991). However, in some C3 and C3-like plants, the H protein is encoded by a small mutigene family (Kopriva and Bauwe, 1995). From sequence comparisons, H-proteins are moderately conserved, and the relatively high mutation rate is probably due to the fact that the H-protein has no known catalytic activity itself (Kopriva et a/., 1996).
In chicken, coupled transcription of the P- and H-protein takes place and since these genes are coordinately expressed under particular conditions, it was suggested that these genes must contain a common regulatory element (Kure eta/., 1991 ).
The T-protein of GDC
The lipoamide cofactor of the H-protein leaves the active site of the P-protein and moves to the active site of the T -protein. The methylene carbon of glycine is transferred to tetrahydrofolate, the a-amino group is lost as ammonium, and the lipoamide is left in the reduced form (Hiraga and Kikuchi, 1980b). A highly conserved region of the T-protein is characterised by the presence of basic residues, which may be involved in the binding of
THF (Bourguignon eta!., 1993 ). In plants, the T -protein plays a strategic role in the
21 Introduction
oxidative photosynthetic carbon cycle since it is responsible for the release of ammonia and
catalyses the first step of the photorespiratory nitrogen cycle (Givan eta!., 1988).
The L-protein of GDC
The reduced lipoamide of the H-protein, resulting from the transfer of glycine, is
oxidised back to lipoamide by an FAD coenzyme bound to lipoamide dehydrogenase (LPDH;
L-protein) with the sequential reduction of FAD and NAD+ (Kim and Oliver, 1990;
Neuburger et al., 1991). The L-protein has been extensively studied in both bacterial and eukaryotic species as a component of several multienzyme complexes: pyruvate
dehydrogenase (PDH), 2-oxoglutarate dehydrogenase (2-0DH), branched-chain 2-oxo acid
dehydrogenase and the GDC (Dickinson and Dawes, 1992; Dickinson eta!., 1986; Guest et al., 1984; Stephens et al., 1983).
Pseudomonas putida has up to three different LPDH's: LPD-glc is required for
PDH, 2-ketoglutarate dehydrogenase and GDC, but LPD-val is specific for the branched chain keto acid dehydrogenase (Sokatch and Burns, 1984; Sokatch et al., 1981; Sokatch et al., 1983). LPD-3 has no clear function other than it can substitute for the LPDH of the 2-
0DH and PDH when the latter is inactive or missing (Burns et al., 1989; Palmer et al.,
1991 ). In humans, there has been no indication of presence of multiple copies of the gene
(Otulakowski et al., 1988), however, there is immunological evidence for two LPDH's in rats (Carothers et al., 1987). In plants, Southern analysis showed LPDH is encoded by a single gene (Bourguignon eta!., 1992). The same LPDH from pea leaf mitochondria is thought to be shared by the mitochondrial enzyme complex, PDH, 2-0DH and GDC
(Bourguignon et al., 1996; Turner et al., 1992b).
Alignment of the deduced amino acid sequence of human LPDH with human erythrocyte glutathione reductase revealed extensive homologies (about 33% identity) throughout the primary sequence. In particular, in the pyrophosphate-binding loop of the
FAD domain, the redox active cysteines and the sequences surrounding the active site found in the C-terminal region of glutathione reductase are exactly maintained in LPDH suggesting
22 Introduction
that this sequence is involved in interactions with the similar disulphide substrates
(glutathione and lipoamide ). There are remarkable similarities in the secondary and tertiary structures among lipoamide dehydrogenases and glutathione reductases in yeast and human
(Carothers eta!., 1989; Otulakowski and Robinson, 1987).
1.3.2 Regulation of GDC in E. coli
Little is known about the molecular mechanisms regulating expression of genes for the GDC in eukaryotes, in spite of its importance in one-carbon metabolism. Most of the available know ledge has come from the study of E. coli.
In E. coli, three components of the glycine cleavage system, the GcvT, GcvH, and
GcvP proteins, are encoded by the gcv operon (Plamann eta!., 1983), induced by glycine
(Meedel and Pizer, 1974; Plamann et al., 1983; Wilson eta!., 1993b), and repressed by purines (Ghrist and Stauffer, 1995; Wilson et al., 1993a). It appears that expression of the gcv operon is regulated in order to balance the cellular requirements for glycine and one carbon units.
Five proteins, the leucine-responsive regulatmy protein (Lrp), the purine repressor protein (PurR), the glycine cleavage activator protein (Gcv A), the glycine cleavage repressor protein (GcvR) and cAMP receptor protein (CRP) have been shown to be involved in regulating expression of gcv operon. Lrp is a global protein regulating expression of numerous genes involved in amino acid metabolism (Calvo and Matthews, 1994) and is required for normal induction of gcv (Stauffer and Stauffer, 1994). Lrp binds to multiple sites upstream of the gcv promoter, suggesting a direct role for Lrp in gcv expression. Lrp may play a primarily structural role by bending the DNA while GcvA functions as the activator protein (Stauffer and Stauffer, 1998). PurR is a negative regulator of many genes involved in nucleotide metabolism, including those of the gcv operon (Wilson eta!., 1993a).
PurR mediates a twofold decrease in gcv transcription in response to purine supplementation and has been shown to bind to the gcv control region.
23 Introduction
GcvA and GcvR, which are gcv-operon specific, work in concert to further regulate gcv expression. In glucose minimal medium, GcvR negatively regulates gcv expression, resulting in low, basal level expression (Ghrist and Stauffer, 1995). In glycine supplemented cultures, repression by GcvR is relieved, and Gcv A activates gcv expression
(Ghrist and Stauffer, 1995; Wilson eta!., 1993b). In purine supplemented culture, both
Gcv A and GcvR are required to modulate a PurR-independent repression of gcv (Ghrist and
Stauffer, 1995; Wilson eta!., 1993a). Therefore, the GcvA protein functions as both an activator and a repressor for the gcv operon expression and the GcvR protein is involved only in the repression of gcv, although its ability to repress is dependent on a functional
Gcv A protein.
It has been shown that gcv expression is altered by changing the ratio of Gcv A and
GcvR. Overexpression of Gcv A leads to constitutive activation of gcv, even in the absence of glycine, while overexpression of GcvR causes super-repression of gcv even in the absence of purines (Ghrist and Stauffer, 1995). It has been shown that glycine and inosine have no effect on the expression of Gcv A and GcvR. Both genes are transcribed constitutively with respect to medium richness and growth phase, and there is no reciprocal regulation between these two proteins (Ghrist and Stauffer, 1998).
Several models were suggested for the regulation of the gcv operon. First, Gcv A homocomplexes may function as activators, while GcvA-GcvR heterocomplexes may function as repressors. Presence of the coregulators, glycine and purine lead to the formation of activator/repressor complexes, respectively. Secondly, GcvR may synthesise the corepressor required for the repressor function of Gcv A. According to this model, in a gcvR mutant, insufficient corepressor would lead to constitutive gcv expression. If GcvR is overproduced, too much corepressor causes super-repression of gcv. Thirdly, GcvR may negatively regulate gcv by modifying the structure of GcvA, changing it from an activator to a repressor in response to the coregulators (Ghrist and Stauffer, 1998).
24 Introduction
Recently, the cAMP receptor protein (CRP) has been shown to be a positive regulator of the gcv operon (Wonderling and Stauffer, 1999). Deletion of the crp gene resulted in a three- to four-fold decrease in gcv expression, and also showed that CRP requires cAMP for regulation of the gcv gene. It appears that CRP plays a role in the regulation of gcv by interfering with repression by GcvR, rather than to activate transcription via interactions with
RNA polymerase.
1.4 Regulation of nitrogen metabolism in S. cerevisiae.
From previous studies, it has been shown that wild-type cells could grow on glycine as sole nitrogen source, whereas cells lacking GDC activity could not (Sinclair and Dawes,
1995). Therefore, the one-carbon and nitrogen metabolism pathways in S. cerevisiae are connected via the GDC and it is relevant to studies characterising control of the GDC that nitrogen metabolism and its regulation be considered.
1.4.1 Introduction
The survival of unicellular organisms such as yeasts in their natural environment is dependent on their ability to adapt rapidly to changes. For example, yeast cells must respond to a constantly changing environment in the availability of nitrogen sources (N-sources) to survive. S. cerevisiae has evolved to selectively utilise high-quality nitrogen sources first when there are more than one N-source available. There are at least two systems to make this selection: short-range and long-range modulation (Cooper, 1982). Short range modulation includes extracellular/intracellular compartmentation. Cells exhibit a 30-fold decrease in their ability to accumulate poor N -sources in the cell after five minutes incubation in a medium containing a good N-source (Cooper and Sumrada, 1983). Nitrogen catabolite repression
(NCR) can be considered as one of the long range modulation events because of the longer time to effect the end result (a reduction in enzyme levels) of repression, although changes in mRNA levels are rapid (Cooper, 1982; Lawther and Cooper, 1975).
25 Introduction
In cells growing in minimal medium, all cellular nitrogen originates from the amino nitrogen of glutamate (88%) or the amide group of glutamine (12%) and the N-source permitting most rapid growth is glutamine, which serves as an excellent source of both intracellular glutamine and glutamate (Magasanik, 1992).
Utilization of ammonia occurs exclusively by its incorporation into glutamate and glutamine. Ammonia can be incorporated into glutamate by NADP+ -linked glutamate dehydrogenase (NADP-GDH) or into glutamine by glutamine synthetase (GS).
NH4+ + NADPH +a-ketoglutarate <--> glutamate+ NADP+ (NADP-GDH)
NH4+ +glutamate+ ATP <--> glutamine+ ADP +Pi (GS)
GS is essential for growth in ammonia minimal medium (Dmin) since mutants lacking this enzyme can only grow when suppled with glutamine (Mitchell, 1985; Mitchell and
Magasanik, 1983). However, NADP-GDH is not essential for growth on Dmin since mutants lacking it can still grow at half the rate of the wild type, due to the combined action of GS and glutamate synthase (GOG AT) (Folch eta!., 1989; Miller and Magasanik, 1990).
Glutamine+ a-ketoglutarate+ NADH --> 2 glutamate+ NAD+ (GOG AT)
GOGAT is also not essential for growth in Dmin and, moreover, a mutant lacking both
NADP-GDH and GOGAT was still able to grow on Dmin, suggesting the existence of another pathway for glutamate synthesis. Recently, a third pathway for glutamate synthesis was found in S. cerevisiae, which is catalysed by an NADP-GDH isozyme encoded by
GDH3 (Avendano et al., 1997). A triple mutant of NADP-GDH, GOGAT and the NAPD
GDH isozyme was a strict glutamate auxotroph.
The amino nitrogen of glutamate provides 88% of the cellular nitrogen requirements
(Magasanik, 1992). To provide the remaining 12% from glutamine, glutamate is broken down to ammonium by the action of NAD-linked glutamate dehydrogenase (NAD-GDH), which is then combined with a second molecule of glutamate via the action of GS.
26 Introduction
glutamate+ NAD+ <-->a-ketoglutarate+ NADH + NH4+ (NAD-GDH)
The only physiological role of NAD-GDH is to provide ammonia for the synthesis of glutamine (Miller and Magasanik, 1990). Therefore, any nitrogen compounds that can be transported into the cell and provide either glutamate or ammonia can be used as a soleN source by the yeast cell. The interconversion of ammonia and glutamate form the interface between biosynthesis and degradation of nitrogenous compounds. Glutamate and ammonia can serve as soleN-source and participate along with glutamine, as the major nitrogen donors in biosynthetic reactions.
Cells grow somewhat slower with either glutamate or ammonia as a sole source of nitrogen. In the former case, cells are deficient in glutamine but not glutamate, since the synthesis of glutamine depends on the generation of ammonia from glutamate by the NAD linked glutamate dehydrogenase. In the latter case, the cells are deficient in glutamate but not glutamine, since the synthesis of glutamate depends on the NADP-linked glutamate dehydrogenase, while the resulting glutamate readily reacts with ammonia to produce glutamine via the GS.
The only product of proline is glutamate and the only nitrogenous product of urea is ammonia (Cooper, 1982), which partly explain why these N-sources are considered as poor
N-sources.
1.4.2 Nitrogen catabolic repression (NCR)
S. cerevisiae cells selectively scavenge nitrogenous compounds from their environment. Successful utilisation of N-sources requires cells to have the capacity to modulate the level of nitrogen metabolising enzymes rapidly and precisely. This is accomplished by NCR, the term given to the physiological process through which transcription of genes encoding proteins needed for the utilization of poorly used (non repressing) N-sources is maintained at low levels when more readily used (repressing) N sources are available.
27 Introduction
The classification of high- and low-quality N-sources is not always easy, and is usually, strain and growth-condition dependant. Cooper (1982) suggested three characteristics for good nitrogen sources, "the compound must enter the cell rapidly, be converted rapidly in as few steps as possible to glutamate and/or ammonia and have no toxic side effects on the cell". For most strains, glutamine and asparagine are good N-sources whereas proline and allantoin are poor ones. Ammonia has been arguably considered a good
N-sources but the presence of ammonia on its own is not thought to be a good N-source
(Cooper, 1982).
In S. cerevisiae, a number of enzymes involved in nitrogen metabolism are coordinately regulated in response to the N-source and responsiveness to NCR is a characteristic of most nitrogen catabolic genes.
1.4.3 Nitrogen regulatory circuit in S. cerevisiae
The control network for nitrogen catabolic enzymes including protease and transport systems of S. cerevisiae is regulated in response to the global nitrogen supply in general and to pathway-specific N-sources in particular. The first and dominant mode of regulation is a global transcriptional response mediated by NCR to the level of available nitrogen. GAT A family proteins Gln3p, Gat! p/Nill p, Dal80p/U ga43p, and Deh I p/Gzf3p/Nil2p are transcription factors responsible for this regulation and the genes encoding them are also highly cross-regulated (Coffman et al., 1997; Daugherty et al., 1993; Soussi-Boudekou et al., 1997). The most straightforward genetic model to describe the coordinated regulation of
NCR-sensitive gene expression is that pairs of positively and negatively acting GATA factors antagonise each other's operation, with the resulting gene expression level ultimately dictated by N-source quality and its influence on transcription supported by Gln3p and Gat! p (Figure
1.4 ).
Transcriptional activation requires Gln3p encoded by GLN3, which is homologous to the metazoan GAT A protein family containing zinc finger motifs which bind to GA T(AIT)A core sequence (UASNTRelements) on DNA (Ko et al., 1991; Minehart and Magasanik, 1991;
28 URE2
Figure 1.4 Nitrogen regulatory network inS. cerevisiae. Four GATA factors and Ure2p arc shown. Gln3p and Gat! p are activators and Dal80p and Deh I p are repressors in the NCR system. Arrows indicate positive regulation and closed bars indicate negative regulation. Thick lines are operational in nitrogen derepression conditions and thin lines in nitrogen repressive conditions. Dotted lines represent reduced transcriptional activation function in good nitrogen source. Introduction
Plumb et al., 1989). Gln3p can bind to single GATAAG sequences, although DNA fragments with multiple GATAAG sequences yielded the highest level of DNA-Gln3p complex (Cunningham et al., 1996). Immunoprecipitation experiments and gel-mobility shift assays of Gln3p interacting with DNA fragments containing UASNTR confirmed this view
(Cunningham eta/., 1996; Minehart and Magasanik, 1991). Similarly, the NIT2 protein of
N. crassa and AREA protein of A. nidu/ans contain GAT A zinc finger domains and up regulate the expression of many genes involved in various nitrogen-catabolic pathways (Fu and Marzluf, 1990; Kudla et al., 1990). It appears that these global nitrogen regulatory proteins are responsible for the majority of the NCR response in A. nidulans and N. crassa
(Perrine and Marzluf, 1986).
Interestingly, a single VASNTR site can function in association with an unrelated site and cognate proteins to mediate transcription in S. cerevisiae. It was shown that when a
UASNTR site functions in combination with an unrelated site, the regulatory response contains characteristics of controls derived from both sites (Rai et al., 1995). Such a combinatorial effect was also found in the mouse EpoR promoter where the integrity of both GAT A-1 and
SP 1 binding sites are required, suggesting a potential cooperation between these two transcription factors in establishing full promoter activity (Zon et al., 1991).
Another regulatory protein in nitrogen catabolism, Dal80p/U ga43p down-regulates the expression of genes of multiple nitrogen catabolic pathway in S. cerevisiae (Daugherty et al., 1993) and binds to a cis-acting element URScATA (GAT AA at its core) (Cunningham and
Cooper, 1993). The deduced sequence of Dal80p contains at least 2 motifs, a leucine zipper and a GAT A zinc finger, both of which are essential for its function (Coornaert et al., 1992;
Cunningham and Cooper, 1991 ).
Both Gln3p and Dal80p bind to sequences containing GAT A at their core. If the 2 proteins compete for the same binding site, together with the fact that DALBO expression requires Gln3p and is autogenously repressed by Dal80p itself (Coornaert et al., 1992;
Cunningham and Cooper, 1991), only one of the competing proteins needs to be regulated
29 Introduction
for NCR since one's regulation is already linked to the concentration of the other
(Cunningham et al., 1994; Minehart and Magasanik, 1991). Northern analysis on expression of numerous genes related to nitrogen catabolism in strains with disruption of
DAL80 or deletion of the GLN3 gene indicates that there is a significant difference between them in either the Dal80p and Gln3p DNA-binding sites or subsequent protein-protein interactions (Daugherty et al., 1993). If the two proteins simply bind to UASNrR homologous sequences, then each of the UASNrR containing genes would be expected to respond to genetic loss of DAL80 and GLN3 products. However, Dal80p does not regulate all genes whose transcription is Gln3p-dependent (Daugherty et al., 1993). An explanation of this observation is provided by studies of the Gln3p and Dal80p DNA-binding sites.
Overall Gln3p-binding requirements are somewhat different from that of Dal80p which binds to some but not all UASNrR elements. A significant difference is that optimal DNA binding by Dal80p requires two GATAA-containing sequences oriented tail to tail or head to tail, 15 to 35 bp apart (Cunningham and Cooper, 1993; Cunningham et al., 1994), whereas Gln3p can bind single GATA sequences (Blinder and Magasanik, 1995; Smart et al., 1996; Svetlov and Cooper, 1997).
There is another repressor system other than Dal80p. Ure2p-mediated regulation, together with Dal80p form a mutually exclusive repression system (Andre et al., 1995).
Gln3p operation is thought to be regulated by Ure2p in response to the quality of N-source availability rather than GLN3 steady state mRNA levels (Coffman et al., 1994). Under nitrogen-repressing conditions, Ure2p disables Gln3p and a mutant lacking Ure2p expresses
Gln3p-activated genes in the presence of good N-sources (Magasanik, 1992). Ure2p probably does not interfere with the binding of the Gln3p to UASNrR sites but rather acts directly on Gln3p to disable its ability to activate transcription (Blinder et al., 1996; Blinder and Magasanik, 1995).
It was shown that NCR-sensitive transcription of some genes is not entirely Gln3p dependent (Andre et al., 1995; Coffman et al., 1995), and during a study of the gln3 ure2
30 Introduction
da/80 triple mutant, there was still NCR-sensitive expression of multiple nitrogen catabolic genes, indicating an additional component of the system (Coffman eta!., 1995). Another
GAIA factor encoded by the N/L/IGATJ gene is responsible for this Gln3p-independent
NCR sensitivity (Coffman eta!., 1996; Stanbrough eta!., 1995). Like Gln3p, Nillp contains a putative GAT A-zinc finger type DNA-binding motif and it was shown that Nillp utilises UASNTR sites to activate the expression of genes (Stanbrough and Magasanik, 1996).
Gln3p and Nil! p also have highly acidic amino-terminal regions, a feature characteristics of transcriptional activators. The lack of such regions in Dal80p and the other repressor, Dehlp is in accord with their role as repressors, rather than activators, of transcription (Stanbrough et a!., 1995).
A fourth GAIA factor, Gzf3p (also called Deh1p/Nil2p) which most closely resembles Dal80p, has the properties of a negative GAIA factor (Coffman eta/., 1997;
Rowen eta!., 1997; Soussi-Boudekou et al., 1997). Gzf3p differs from the other negative regulator Dal80p in that, while Dal80p/Uga43p is active specifically under N-derepression conditions, Gzf3p exerts its negative regulatory function specifically in good N-sources with a major effect to repress Gatlp/Nillp-dependent transcription (Soussi-Boudekou eta!.,
1997). The transcriptional activator, Gat1p is also subject to two negative regulatory systems: under N-derepressing conditions, negative control is effected by Dal80p, but when a good N-source is available (when DALBO is not expressed), Nillp-expression is subject to
N-repression partially dependent on Gzf3p. It was also shown that Gzf3p can potentially repress Gln3p-dependent expression to a significant extent, but this effect is visible only in the absence of Uga43p (Soussi-Boudekou eta!., 1997). Dehlp and Dal80p share two structural features, the leucine-zipper and zinc-finger motifs but Deh1p is twice as large as
Dal80p (Coffman et al., 1996; Coffman eta!., 1997; Soussi-Boudekou eta!., 1997). Both
Dal80p and Deb I p are capable of homodimerisation and forming DalSOp-Deb I p heterodimeric complexes, but Gln3p and Nill p/Gat I p lack the C-terminal leucine zipper motif required for the normal operation of Dal80p (Svetlov and Cooper, 1998).
31 Introduction
Yeast possesses a network of cross- and auto-regulation among the GAT A factors
(Figure 1.4). Gln3p appears to occupy a special position in this regulatory network, being the only GAIA factor whose gene is not regulated by N-sources (NCR-insensitive).
Instead, Gln3p operation is principally regulated through its DNA binding and/or its ability to support transcriptional activation (Cunningham et al., 1996; Minehart and Magasanik, 1991).
GAT/ expression is Gln3p-dependent, Dal80p-regulated, and in some strains and growth conditions, Deh1p and Gat1p regulated. DALBO expression is Gln3p- and Gatlp-dependent as well as Dal80p- and Dehlp-regulated. Each of Gln3p and Gatlp alone can activate the
DALBO gene and the contribution of the two factors are additive. DEHI expression also requires at least one of the two positive regulators, but is the least dependent of the four
GAT A factor genes on Gln3p, and is partially Gat! p dependent and highly regulated by
Dal80p and Dehlp (Coffman et al., 1997; Rowen et al., 1997; Soussi-Boudekou et al.,
1997). A major role of these regulatory loops is to ensure the proper balance between the concentrations of the four GAIA factors. This balance is tightly regulated according toN supply so that the factors are synthesized at limited but different levels, and mutually exclusively. This results in the ability of cells to respond finely to a variety of disparate environmental stimuli smoothly by passing from one response to another.
From various observations, however, it has been suggested that other nitrogen regulatory factors remain to be discovered. It has been reported that U re2p can repress the expression of PUTJ in cells lacking Gln3p (Xu et al., 1995), and it was also observed that in gzf3 mutant cells, Nil! p still partially responds to N-repression, especially under the optimal
(combination of two good N-sources) N-supply conditions. In addition, some nitrogen pathway genes remain partially sensitive toN-repression in cells lacking Ure2p, Gzf3p, and
Uga43p (Soussi-Boudekou et al., 1997).
Recently, a co-activator of nitrogen-regulated transcription in S. cerevisiae was identified (Soussi-Boudekou and Andre, 1999). Adalp is required for full expression of many nitrogen metabolic genes and is involved in Gln3p- and Nill p-dependent transcription.
32 Introduction
Ada 1p also plays a role in repression of some nitrogen metabolic genes, MEP 1, MEP 3 and
CARl.
1.5 The mechanism of transcriptional regulation
Transfer of information from DNA to protein is mediated by mRNA. The initiation
stage of mRNA synthesis in eukaryotic nuclei is a major point of regulation in the control of
both positive and negative effectors. In eukaryotes, transcriptional regulation involves
interactions between DNA elements, transcription factors, cofactors, basal transcription
machinery and the chromatin/chromosomal environment.
Eukaryotic promoters can be divided into core elements and regulatory elements.
Core, or basal promoter elements define the site for assembly of the transcription preinitiation
complex (PIC) and include a TATA sequence for binding of the TAT A-binding protein
(TBP), located 40 to 120 bp upstream of the transcription start site (in S. cerevisiae) and an
initiator sequence (lnr), encompassing the start site (Weis and Reinberg, 1992). Regulatory elements are gene-specific sequences which serve as binding sites for transcriptional
activators and repressors and control the rate of transcription initiation.
There are three classes of transcription factors which associate directly or indirectly
with DNA elements: general transcription factors (GTFs), transcriptional cofactors, and
gene-specific transcription factors. Transcriptional cofactors (coactivators and corepressors),
also known as mediators or adaptors, are distinct from the GTFs in that they are dispensable
for basal-level transcription in vitro and distinct from activators in that most do not directly
bind DNA and none appear to bind DNA in a sequence-specific manner. In some cases, they facilitates chromatin remodelling (Hampsey, 1998).
1.5.1 RNA polymerase II general transcription machinery
Yeast RNA polymerase II (RNA pol II) is composed of 12 subunits encoded by RPB
genes (Woychick and Young, 1994), and its unique feature is the presence of a carboxy-
33 Introduction
terminal repeat domain (CTD) which is highly conserved among eukaryotes with varying repeat length. RNA pol II with an unphosphorylated CTD enters the PIC preferentially. whereas one with a phosphorylated CTD is found in the elongation complex (Dahmus,
1996), implicating conversion of RNA pol II from a form involved in promoter recognition to an elongation competent form (Lu eta!., 1991; O'Brien et al., 1994). The CTD has also been implicated as a platform for the assembly and recruitment of factors involved in pre mRNA processing (Steinmetz, 1997).
Stepwise assembly model of PIC
In vitro, RNA pol II cannot initiate promoter-specific transcription alone and requires an additional set of proteins called the general transcription factors (GTFs), and a stepwise assembly model of the PIC has been suggested (Figure 1.5) (Buratowski eta!., 1989; Van
Dyke eta!., 1988). This defined-order assembly is guided by the TATA box, the DNA sequence recognised by the TBP, either alone or with the TFIID multienzyme complex containing TBP and TI3P-associated factors (TAFs). The other factors are then recruited sequentially into the initiation complex in the following order: IIA, liB, IIF with RNA pol
II, liE, IIH, and III (Buratowski, 1994; Zawel and Reinberg, 1992). The polypeptide components of RNA pol II and most of these GTFs are well conserved among eukaryotes from yeast to human. This stepwise assembly mechanism proposed from in vitro studies provide a guide to protein-protein interactions that are likely to be a subset of the more complex interactions that occur at promoters in vivo.
TBP is a universal transcription factor, required for initiation by all three eukaryotic
RNA polymerases (Hernandez, 1993). TBP was identified as a subunit ofTFIID (Pugh and
Tjian, 1992) and is an essential transcription factor that affects promoter recognition which is a critical determinant of transcriptional activation (Lee and Struhl, 1995). The association of
TFIID with promoter DNA nucleates the initiation process and is the only step in GTF assembly driven entirely by protein-DNA interactions. All subsequent steps entail recognition of prefonned nucleoprotein complexes.
34 ChromatiD TATA lnr
~ Chromatin remodelling machinery + Activators
TATA lnr
Holoenzyme Model Stepwise Assembly Model
TRIA l B TFIIB
RNA polli THilF
RNA pol II Holoenzyme ' J: ::~ TFIIJ
Figure 1.5 Models for the transcription preinitiation complex (PIC) fonnation on TATA containing promoters. A holoenzyme model and the stepwise assembly model are compared. Disruption of the repressive effect of nucleosomal structure can be achieved by chromatin remodelling machineries aided by activators (section 1.5.2). In the holoenzyme model, a preassembled subset of GTFs and RNA pol II binds to the promoter in a single step. Most studies showed neither TBP nor TAID seems to be a component of holoenzyme. In the stepwise assembly model, TAID first binds the TATA element, followed by TRIA and TAIB (section 1.5.1). TAIF escorts RNA pol II to the promoter and the PIC formation is completed by the binding of TAlE, THIIJ and TAIH. Introduction
It was demonstrated that TBP induces kinks at both ends ofTAT A elements, partially unwinding and bending the DNA 80' toward the major groove (Kim et al., 1993a; Kim et al., 1993b), which may increase the proximity of proteins bound on either side of TBP. A critical role of TBP in transcriptional activation was implied by direct contact between the activation domains of many gene-specific activators and TBP (Nikolov and Burley, 1994).
Transcriptional activators enhance the kinetics of TBP recruitment, which is a slow and potentially rate-limiting step in transcriptional activation (Klein and Strub!, 1994), and enhance the formation or stability of the TBP-TAT A complex at certain promoters in vivo
(Arndt et at., 1995).
Although TBP is sufficient for promoter recognition and subsequent assembly of other factors into a functional PIC, transcriptional activation in some systems is observed only with the multisubunit TFIID complex (Hoffman eta!., 1990; Pugh and Tjian, 1990), which led to the discovery of the TBP-associated factors (TAFs). TAFs were proposed to function in relaying information from activators to the core transcriptional machinery.
However, TAFs are not generally required for activation in yeast (Tansey and Herr, 1997), thus they appear to be critical coactivators of only a subset of genes.
The primary role of TFIIB is to physically link TFIID at the promoter with the RNA pol II I TFIIF complex and thus determine the distance to the transcription start site (Li et at.,
1994; Pinto eta/., 1992). TFIIB has also been implicated as the direct target of many gene specific transcriptional activators, leading to the proposal that certain activators stimulate transcription by TFIIB recruitment (Lin et al., 1991; Roberts eta/., 1993). TFIIB engages in an intramolecular interaction that leads to an activator-induced conformational change, allowing assembly of the PIC (Roberts and Green, 1994 ).
Once TFIID and TFIIB have assembled at the promoter, RNA pol II can enter the preinitiation complex escorted by TFIIF (Bengal et al., 1991; Bradsher et al., 1993; Flores et a/., 1989). TFIIF probably does not play a significant role in promoter selectivity but
35 illlroduction
contributes to PIC stability which is required for specific initiation at all promoters tested in yeast (Henry et al., 1992).
TFIIA can join the complex at any stage after TFIID binding and stabilise the initiation complex through interactions with TBP (Imbalzano et al., 1994). TFIIA is located exclusively upstream of the TAT A and is unlikely to contact other GTFs that bind downstream of the TAT A. Only a very small region of TFIIA interacts with TBP and DNA, which leaves a large surface area on TFIIA available to interact with other factors (Geiger et al., 1996; Tan et al., 1996). Indeed, TFIIA has shown to interact with specific transcriptional activators (Ozer eta/., 1994; Yokomori eta/., 1994 ).
The addition of TFIIE and TFIIH to the preinitiation complex completes the assembly process and renders the polymerase competent to initiate transcription. TFIIE has been implicated as the direct target of certain gene-specific transcriptional activators (Sauer eta!.,
1995a; Zhu and Kuziora, 1996). Structure-function analysis indicates that TFIIE might act as a checkpoint for formation of the PIC via its control of TFIIH recruitment and activities
(Ohkuma et al., 1995).
TFIIH is the only GTF with known enzymatic activities which include DNA dependent ATPase (Roy et al., 1994), helicase (Schaeffer et al., 1993), and a CTD kinase
(Feaver et al., 1991; Lu et a/., 1992). TFIIH performs critical roles at both initiation and post-initiation stages of transcription (Dvir eta!., 1996; Holstege et al., 1996). The transition from transcription initiation to elongation is presumably mediated by CTD kinase (Usheva et al., 1992) as is the transition from very early elongation complexes to stable elongation complexes (Dvir et al., 1997). Therefore, TFIIH performs multiple roles in transcription, affecting the steps before, during, and immediately after initiation.
Transcriptional regulation can be mediated during any of the steps of initiation complex assembly. Efficient transcription also requires one or more gene-activator proteins, which are usually bound to DNA upstream of theTAT A box. For at least some genes, an
36 Introduction
additional set of proteins (transcriptional cofactors) which associate with RNA pol II is
required.
RNA pol II holoenzyme
Several GTFs are known to associate with RNA pol II in the absence of DNA, to
form a large complex that is commonly termed the "RNA pol II holoenzyme" (Conaway and
Conaway, 1993). It is no surprise that GTFs and RNA pol II can associate away from the
promoter since numerous GTF-GTF and GTF-pol II interactions have been reported. The
holoenzyme is highly stable in the absence of DNA, and is capable of efficient initiation when
supplemented with TBP and TFIIE (Koleske and Young, 1994 ), suggesting that the
holoenzyme is recruited to promoters at which TFIID is already bound (Figure 1.5). The
RNA pol II holoenzyme contains the "core" RNA pol II, a subset of the basal transcription
factors (such as TFIIB, TFIIE, TFIIF, and/or TFIIH), nine SRB (suppressor of RNA
polymerase B) proteins, as well as other known (such as Galli p, Sin4p, Rgr I p, and Rox3p)
and unknown proteins.
The existence of the holoenzyme was inferred by convergence from genetic and
biochemical approaches. SRB proteins (Srbps) identified by a genetic approach (Liao et al.,
1995; Nonet eta/., 1987; Thompson eta!., 1993) were present in the RNA pol II
holoenzyme containing roughly equimolar of RNA pol II, TFIIF, TFIIB and Srbps (Koleske
and Young, 1994).
Concurrently, a search for protein factors that would allow a purified yeast
transcription system to respond to acidic activators led to the purification of a multisubunit complex, termed a "mediator" (Kim eta!., 1994). Transcription in a crude cell-free system was stimulated by activators, but on reconstitution with purified transcription proteins, the stimulatory effect was lost (Flanagan eta!., 1991; Flanagan eta!., 1992; Meisterernst et al.,
1991). The effect could be restored by the addition of novel protein factors, termed mediators which are involved in in vivo and in vitro transcriptional activation. It is presumed that they play an intermediary role between activators and the basal apparatus by interacting
37 Introduction
with the CTD of RNA pol II (forming RNA pol II holoenzyme) in vivo (Bjorklund and Kim,
1996). It became apparent that the mediator complex and the SRB complex are the same structure and it is often called the Srb/mediator complex. Srb/mediator components appear to confer both positive and negative effects on gene expression, suggesting that the
SRB/mediator of "activation" might be more appropriately termed the Srb/mediator of transcriptional "regulation" (Li eta!., 1995).
The CTD of RNA pol II can be viewed as an antenna serving as an interaction site for multiple Srb/mediator complex, GTFs and additional unidentified components of the pol II holoenzyme for regulatory information and the Srb/mediator complex as a signal-processing device, integrating positive and negative input, transducing them back to the polymerase for control of transcription. It has also been suggested that the holoenzyme transduces activating or repressing signals that alter the CTD phosphorylation state via the holoenzyme-specific polypeptides (for example, Kin28p/Srbl0p) or TFIIH and consequently stimulate or inhibit promoter clearance and chain elongation (Akhtar eta/., 1996; Myers et al., 1998; Valay et al.,
1995). Furthermore, the holoenzyme may contain a CTD phosphatase that is important for transcription termination and other proteins involved in RNA processing (McCracken et al.,
1997).
There are two forms of RNA pol II holoenzyme in yeast cells. One containing Srbps
(Koleske and Young, 1994; Thompson et al., 1993) and the other with Paf!p and Cdc73p but without Srbps (Shi eta!., 1997; Wade eta!., 1996). These are thought to transcribe overlapping subsets of genes because the SRB genes are essential and affecting transcription of most genes (Thompson and Young, 1995), while Paflp and Cdc73p, in addition to the shared components Galli p, Sin4p and Rgr I p are all non-essential and appear to affect only a subset of transcripts (Li et al., 1995; Shi et al., 1997; Suzuki eta!., 1988). The functional overlapping nature of the two complexes is based on the fact that expression of some genes is affected by mutations in either complex.
38 Introduction
In contrast to TAFs (components ofTFIID), which appear to function as coactivators in a gene-specific manner in yeast, the Srb/mediator appears to play a more general role in transcriptional activation. All of the SRB genes are essential for normal yeast-cell growth
(Koleske and Young, 1994; Thompson et al., 1993), and at least some components of
Srb/mediator complex are essential for in vitro transcription in most, if not all, class II genes
(Koleske and Young, 1995). In Drosophila, TAFs (but not mediators) enable a response to activators for the stimulation of transcription. Multiple activators exert their synergistic effects by recruiting TFIID to promoters, accelerating the first step in PIC formation (Sauer et a/., 1995b). Although TAFs can also function as coactivators in yeast (Poon et al., 1995;
Reese et al., 1994), a TAP-independent pathway has also been described (Moqtaderi et al.,
1996; Walker eta/., 1996), suggesting T AFs play more specialised roles in transcription in yeast. Therefore, the response to activators in the yeast system is effected via the
Srb/mediator complex in the absence ofT AFs, while the response in the Drosophila is brought about by a T AF-TBP complex in the apparent absence of mediators (Verrijzer and
Tjian, 1996). In humans, activation requires both TAFs and positive co-factors (Kaiser and
Meisterernst, 1996), raising the possibilities that mediators and T AFs function in concert.
For example, the general co factors stimulate transcription in the presence of the human RNA pol II holoenzyme, which is associated with mediators (Chao et al., 1996).
In the RNA pol II holoenzyme model, two major regulatory steps for activated transcription are the recruitment of TFIID to the promoter and association of the holoenzyme with this complex (TFIID-DNA complex) (Stargell and Struhl, 1996). The rate-limiting step of TFIID-promoter complex formation in transcriptional activation has been described in the previous section. A second rate-limiting step in transcriptional activation involves the recruitment of the holoenzyme. As with artificial recruitment of TBP (TFIID), recruitment of the holoenzyme bypasses the requirement for an activator (Barberis et al., 1995).
Furthermore, the SRB-containing holoenzyme responds to transcriptional activators in vitro, thus containing components necessary and sufficient for responding to transcriptional
39 Introduction
activators (Kim et a/., 1994; Koleske and Young, 1994). Considerable genetic and biochemical evidence indicate that activators recruit the RNA pol II holoenzyme to promoters through interactions with the Srb/mediator complex (Hengartner et al., 1995; Koh eta/.,
1998; Myers et al., 1998).
The RNA pol II holoenzyme may also mediate alterations in chromatin structure that enable the initiation complex to bind to a promoter (Mizzen et al., 1996; Wilson et al., 1996), as it has been shown that the recruitment of the RNA pol II holoenzyme was sufficient for chromatin remodelling (Gaudreau et al., 1997) which is discussed below.
1.5.2 Chromatin structure and transcription
The transcriptional machinery involved in regulation must contend with chromatin, the dynamic matrix of histone and nonhistone proteins with which the DNA template is associated. Chromatin is intrinsically involved in the regulation of nuclear processes, especially transcription. The primary unit of chromatin organization is the nucleosome which consists of about 146 bp of DNA wrapped around two copies each of the core histone proteins H2A, H2B, H3, and H4 (Luger et al., 1997). This can prevent the improper activation of repressed genes by impeding the access of transcription factors to their target site, and thus is a requirement for appropriate gene expression. An early step in gene activation involves an alteration (remodelling) of nucleosome structure at active promoters, which allows binding of transcription factors.
Chromatin remodelling appears to be a prerequisite for transcriptional activation
(Gaudreau et al., 1997; Stafford and Morse, 1997; Wong et al., 1997) and in some cases, chromatin remodelling of a promoter by activator binding is necessary, but not sufficient for productive transcription (Orphanides et al., 1998). Studies with the PH05 and GAL genes in yeast showed that the binding of an activator to its recognition site is not necessarily enough to induce remodelling (Stafford and Morse, 1997; Svaren and Horz, 1997). It has been demonstrated that RNA pol II holoenzyme recruitment can bypass the requirement of an activation domain (Gaudreau eta!., 1997), perhaps nucleosome remodelling machines such
40 Introduction
as TAFs, SWI/SNF-like chromatin remodelling factors, or HAT (histone acetyl transferase) recruited to the promoter via association with the holoenzyme provide the necessary
functionality. Therefore, it seems that activators are involved in recruiting additional large
molecular complexes that assist in the remodelling of chromatin.
Two models were suggested for the role of the nucleosome remodelling machinery in
transcription (Barberis and Gaudreau, 1998). First, it may work constitutively throughout the chromosomes to facilitate the transition between transcriptionally non-permissive and permissive forms of nucleosomes, which are in equilibrium. In the presence of activators,
the recruited RNA pol II holoenzyme complex and the nucleosome remodelling machinery cooperate at the particular region of the chromosome to shift the equilibrium in favour of the transcriptionally permissive form. Secondly, nucleosome remodelling machinery might be generally inefficient unless brought to promoters through interaction with some component of the RNA pol II holoenzyme which is recruited by DNA-bound activators (Wilson et al.,
1996). However, these two models do not consider whether activators themselves need remodelling machines for binding to their recognition sequences. It was shown that activators can indeed bind nucleosomal DNA in vitro in the absence of remodelling machines, although binding affinities clearly increase when nucleosomes are remodelled or disrupted
(Kingston, 1997; Polach and Widom, 1995).
A direct link between chromatin function and acetylation was established by the discovery that coactivator complexes required for transcriptional activation function as histone acetyltransferases (HAT) (Brownell et al., 1996; Kuo eta/., 1998; Ogryzko et al.,
1996), while corepressors containing histone deacetylase activities confer transcriptional repression (Hassig eta!., 1997; Wong et al., 1998).
Histone acetylation has been associated with the interaction of non-histone proteins with his tones (Edmondson eta/., 1996), histone deposition and nucleosome assembly (Roth and Allis, 1996), higher order packing of chromatin (Hendzel eta!., 1998) and the transcriptional activity of cellular chromatin (Turner, 1998). The acetylation of his tones is
41 Introduction
thought to direct their dissociation from the DNA and/or to play a role in loosening
internucleosomal interactions, which leads to chromatin opening and gene activation by
increased accessibility to transcription factors (Luger et al., 1997; Wade et al., 1997).
Histone acetylation states are dynamic, which provides an attractive mechanistic foundation
for the reversible activation and repression of transcription (Wade et al., 1997; Wolffe,
1997). The steady state level of acetylation of histones is a balance between the action of
HAT and histone deacetylase. In concert with the HAT activities, many systems are found to
employ antagonistic histone deacetylation activities in the regulation of gene expression
(Hassig et al., 1997; Heinzel et al., 1997; Kadosh and Struhl, 1997; Nagy et al., 1997).
Recruitment of a histone deacetylase by its interaction with a corepressor results in the
transcriptional repression of the specific promoter presumably via the reduction in the local
level of chromatin acetylation.
These histone acetylase/deacetylase activities are often found to be associated with
large multisubunit protein complexes and contain known regulators of transcription. The functional importance of HAT was highlighted by the discovery of two Gcn5p-containing complexes in vivo, the Ada complex and SAGA (composed of Spt, Ada and GenS subunits)
(Grant eta/., 1997). Gcn5p was shown to interact directly or indirectly through Ada2p with a number of activators, and the connection between transcriptional activation and acetylation was strengthened by the discovery that these proteins possess HAT activity (Kuo et al.,
1998; Pollard and Peterson, 1997).
The ability of transcription factors to recognise their binding sites within nucleosomes is inherent. The intrinsic variance of different transcription factors binding to their sites in the chromatin environment is likely to be the functional basis for the hierarchy of gene regulation in eukaryotes. This can be further complicated by additional protein-protein interactions, or the co-operative binding of different factors to their recognition sites within the nucleosome
(Li et al., 1997).
42 Introduction
Complexes have been described that facilitate transcriptional activation by affecting nucleosome structure but which do not catalyse histone acetylation. For example, a large multisubunit complex named the SWI/SNF complex is involved in the chromatin remodelling process of a small subset of genes in yeast (Cote eta!., 1994). SWVSNF is a high-affinity
DNA binding complex (Quinn eta/., 1996), whose binding to the face of nucleosomes would allow contact with all four core histones. It might be then act to alter the histone contacts with DNA, and destabilise higher order chromatin structure (Wolffe and Hayes,
1999). There are models explaining why the yeast SWVSNF complex is required for full transcriptional activity of some, but not all promoters (Kadonaga, 1998). The complex is targeted to a selected subset of promoters, which is achieved by interactions between the
SWI/SNF complex and sequence-specific transcription factors and/or by sequence-specific
DNA binding by the SWI/SNF itself. Alternatively, it could be that only weak promoters require SWVSNF function for full activity (Burns and Peterson, 1997).
Other yeast chromatin-remodelling complexes include the RSC complex which is functionally distinct from the SWVSNF complex (involved in cell-cycle progression), and is abundant and essential for viability (Cairns eta!., 1996; Cao eta/., 1997) and STP/SIN proteins which repress transcription by formation of inactive chromatin (Winston and
Carlson, 1992). Many of the genes that show SWI/SNF dependence also demonstrate dependence on HAT, GenS (Pollard and Peterson, 1997), and similarly for SAGA,
SNF/SWI and SRB/mediator complexes (Roberts and Winston, 1997). Therefore, multiple, partially redundant and interdependent mechanisms act to relieve the chromatin repression of a given promoter.
1.5.3 Relevant transcription factors
General control of amino acid biosynthesis
Starvation of a single amino acid results in increased transcription of genes encoding enzymes of different pathways involved in amino acid biosynthesis (Hinnebusch, 1992;
Hinnebusch, 1988), in purine synthesis (Mosch eta/., 1991) and in aminoacyl-tRNA
43 Introduction
synthesis (Mirande and Waller, 1988). InS. cerevisiae, this regulatory network is known as general control of amino acid biosynthesis and is controlled by the transcriptional activator
Gcn4p which binds to specific DNA sequences called Gcn4p-responsive elements (GCRE),
which are present upstream of more than 50 target genes (Hinnebusch, 1992). The GCRE is well defined and consists ofTGA(C/G)TCA (Oliphant et al., 1989).
Gcn4p is a member of the basic leucine zipper (bZIP) protein family (Ellenberger et at., 1992) and binds directly as a homodimer to a conserved regulatory region of its target genes (Hope and Stmhl, 1986). The bZIP motif consists of two independent subdomains, a basic region contacting DNA and an adjacent one with heptad repeats of leucines (leucine zipper) mediating dimer formation (Vinson et al., 1989). bZIP proteins bind dyad-symmetric binding sites as dimers with distinct dimerization properties. For example, Gcn4p forms
homodimers, Jun dimerizes with itself or with Fos, and Fos only dimerize with Jun (Hope and Stmhl, 1987; Kouzarides and Ziff, 1989; O'Shea et al., 1989).
When cells are grown under conditions of amino acid limitation, an elevation in the cellular amount of Gcn4p is accomplished through an increased translation of GCN4 mRNA by a regulatory mechanism involving phosphorylation of translation initiation factor elF2 by the protein kinase Gcn2p (Hinnebusch, 1994). A scanning-reinitiation model for this translational control has been proposed (Figure 1.6): the four short upstream open reading frames (uORFs) in the leader sequence of the GCN4 mRNA prevent efficient translation initiation at the GCN4 start codon under non-starvation conditions by restricting the progression of scanning ribosomes from the cap site to the GCN4 initiation codon
(Hinnebusch, 1984; Thireos et a/., 1984 ). Consistent with this model, eliminating the start codon of all four uORFs resulted in a high level of GCN4 expression even in non-starved cells (Mueller and Hinnebusch, 1986).
When amino acid are abundant, ribosomes that have initiated translation at the GCN4 uORFs dissociate from the mRNA before they are able to reinitiate at the GCN4 open reading frame. The first uORF (uORFl) is the least inhibitory (Mueller and Hinnebusch, 1986),
44 Introduction
ribosomes translate the uORF I and reinitiate at one of the remaining uORFs in the mRNA leader. However, translation is forced to reinitiate at u0RF2-4 because ribosomes rebind the e!F2/GTP/Met-tRNAMET ternary complex (GTP bound form of e!F2 that delivers charged initiator tRNAMET to the ribosomes) before reaching the uORF4, after which they dissociate from the mRNA. Therefore, reinitiation at the inhibitory uORF2-4 precludes subsequent reinitiation at GCN4 (Hinnebusch, 1997).
Under conditions of amino acid starvation, ribosomes that have translated the uORFI are able to reinitiate further downstream at the GCN4 coding sequences. The failure to reinitiate at uORFs 2 to 4 results from the reduction of the elF2/GTP/Met-tRNAMET ternary complexes concentration due to the phosphorylation of elF2a in starved cells (Dever et al.,
1992). Lacking initiator Met-tRNAMET, ribosomes cannot recognise the AUG codon at uORFs 2-4 (Cigan et al., 1988). Consequently, many ribosomes bypass the inhibitory uORFs 2-4, and most of these ribosomes will bind the ternary complex while scanning between u0RF4 and GCN4, enabling them to reinitiate at the GCN4 start codon (Abastado et al., 1991).
This regulation depends on the phosphorylation of the a-subunit of elF2 (elF2a) by the protein kinase Gcn2p (Dever et al., 1992), which reduces the amount of active initiation factor elF2 available for ternary complex formation. Gcn2p is the sensor for the degree of uncharged tRNAs which appear to be the activating ligand for Gcn2p (Wek eta/., 1995).
Gcn2p consists of anN-terminal kinase domain and a C-terminal tRNA synthetase domain
(Roussou et al., 1988; Wek eta/., 1989). Under amino acids limitation, binding of uncharged tRNAs to the tRNA synthetase domain of Gcn2p results in activation of the neighbouring kinase domain. The phosphorylation of elF2a by Gcn2p inhibits the complex exchange factor elF2B, which catalyses the exchange of bound GDP for GTP on elF2 to deliver initiator tRNAMET to the ribosome after each round of initiation (Merrick, 1992;
Voorma eta!., 1994). The resulting reduction of active elF2, and subsequently low ternary
45 Introduction
complex fonnation, allows ribosomes to scan past the uORFs 2-4 without rebinding charged initiator tRNAMET (Figire 1.6).
Uncharged tRNA
GCNI GCN2 .-__ GCN20
elF-2 I GDPL. elF-2-P
(··················..L.... elF-2B IGTP elF-2 I GTP (·························· tRNAMET
elF-2 I GTP I tRNAMET ternary complex (low level) ~
GCN4
Activation tof genes for amino acid biosynthesis
Figure 1.6 Translational control of GCN4 by the protein kinase Gcn2p. Under amino acid starved condition, phosphorylation of clF-2 by Gcn2p inhibits the fonnation of c!F2/GTP/Met-tRNAMET ternary complexes. In such condition, ribosomes scan through the uORFs 2-4, and can translate GCN4. When amino acids are abundant, dotted lines are highly active, level of ternary complex formation is high, which make ribosomes reinitiate translation of inhibitory uORFs 2-4 before reaching GCN4. Numbered boxes indicates uORFs 1-4.
When Gcn2p was replaced by mammalian kinases, GCN4 translation was stimulated independently of amino acid levels (Dever eta!., 1993), indicating that increased phosphorylation of elF2a mimics the amino acid starvation condition. Also, in starved cells,
GCN2 expression itself is not increased but function of Gcn2p is stimulated (Wek et al.,
46 Introduction
1990). The Gcnlp and Gcn20p are also required for activation of Gcn2p kinase function since mutations that inactivate these proteins reduces or abolish phosphorylation of elF2a by
Gcn2p (Marton et a!., 1993; Vazquez de Aldana eta!., 1995). In fact, there are at least 10 additional genes involved in the complete transmission of the amino-acid starvation signal from Gcn2p to GCN4 translation regulation. They show either a Gcn- (general control non repressed) or Gcd- (general control derepressed) phenotype (Niederberger et al., 1986).
A gcn2 mutant is unable to initiate the GCN4 translational regulation. gcd mutations are genetically epistatic to a gcn2 defect, leading to constitutively derepressed translation of
GCN4 mRNA, whereas a deletion of GCN4 is epistatic to a gcd mutation, suggesting a linear signal transduction pathway (Hinnebusch, 1992).
Baslp/Bas2p
Bas l p transcription factor which binds to the sequence TGACTC is required for the expression of all the genes repressed by adenine studied so far and all the reported Bas 1p responding genes are repressed by adenine (Daignan-Fornier and Fink, 1992; Denis and
Daignan-Fornier, 1998; Springer eta!., 1996; Stotz et al., 1993; Tice-Baldwin eta!., 1989).
The TGACTC binding site seems necessary but not sufficient for activation in the presence of
Bas 1p since expression of several genes carrying the TGACTC motif in their promoter is not affected by mutation of BASI (Denis et al., 1998; Denis and Daignan-Fornier, 1998). This
Myb-like DNA-binding protein was shown to interact with Bas2p to activate H!S4 and various AD£ genes thus mediating cross-pathway regulation between purine and histidine biosynthesis in yeast (Daignan-Fornier and Fink, 1992; Tice-Baldwin eta!., 1989; Zhang et al., 1997). These studies also showed that all the genes activated in the presence of BASI require BAS2 for optimal expression.
The BAS2 gene encodes a homeodomain DNA-binding protein, and was initially identified as PH02, a gene required for derepression of the inducible acid phosphatases encoded by the PH05 and PHOil genes by the cooperative binding with Pho4p, a basic helix-loop-helix protein (Barbaric eta!., 1996; Kramer and Andersen, 1980). Bas2p has also
47 Introduction
been shown to bind to the promoter of the TRP4 and H 0 genes, and in the H 0 gene, it interacts with the Swi5p zinc finger protein (Braus eta/., 1989; McBride eta/., 1997).
However, no clear consensus binding site for Bas2p has been defined. While Bas2p binds on its own to DNA in vitro, it has shown in vivo to require other transcription factors for transcriptional activation (Arndt eta/., 1987; Daignan-Fornier and Fink, 1992; Tice-Baldwin et al., 1989). Therefore it appears that Bas2p is a pleiotropic effector that can interact with at least three other gene-specific transcription factors.
A model has been proposed for regulation by Bas I p and Bas2p in response to adenine. The activation function of Bas I p was stimulated in an adenine-regulated fashion in the presence of Bas2p. In contrast, Bas2p was found to activate transcription independently of both BASI function and the adenine levels in the media. Moreover, the DNA-binding activity of Bas2p was not needed to support activation by Baslp, indicating that Baslp recruits Bas2p and their physical interaction is critical for generation of a potent transcriptional activator complex, which unmasks a latent activation function in Bas I p at the
AD E gene promoters (Zhang et al., 1997). These results also suggest that a purine nucleotide directly or indirectly modifies the ability of Baslp to activate transcription, possibly by affecting its interaction with Bas2p. It was shown that the regulatory signal is not adenine but rather ADP or a derivative of ADP (Guetsova et al., 1997).
However, at the PH05 promoter, the mechanism of action of Bas2p seems different from ADE genes since its DNA binding is critically required for Bas2p function (Barbaric et a/., 1998). Also, it was shown that mutation in the Bas2p DNA-binding domain almost entirely abolished activation by promoter elements of HIS4 and HO promoter (Justice eta/.,
1997).
The effect of extracellular adenine and the role of the transcriptional activator Bas I p on expression of the yeast genome was assessed recently by two-dimensional analysis of the yeast proteome (Denis et al., 1998). It was shown that de novo purine synthesis and some of histidine biosynthesis (HISJ, HIS4 and HIS4 expression) are coregulated. Furthermore,
48 Introduction
it was shown that there is cross-pathway regulation between purine and pyrimidine synthesis, although it does not appear to be a direct effect of Bas 1p or Bas2p (Denis eta/,,
1998),
1.6 Aims of the thesis
The ultimate aim of this study is to understand how the transcriptional regulation of the synthesis of enzymes involved in one-carbon metabolism is achieved in the eukaryote, S. cerevisiae. It is critical toward identification of all genes that are co-regulated by this control system.
One-carbon metabolism is crucial to various cellular processes (Figure 1.2) and because of its nutritional and clinical importance, there have been numerous biochemical studies on the roles of folates and flow of one-carbon units. Genetic studies of yeast using mutants of one-carbon metabolic enzymes have aided in the understanding of roles and functions of each enzyme (section 1.2).
However, little is known about the regulation of one-carbon metabolism in eukaryotes at the molecular level. Although transcriptional control of one-carbon metabolism in E. coli has been studied in detail (section 1.3.2), metabolic pathways and its regulation in eukaryotes are fundamentally different. This study uses the yeastS. cerevisiae as a model organism. The metabolic pathways and transcriptional controls in yeast and higher eukaryotes are essentially identical, but advanced molecular genetic techniques available in yeast combined with its rapid growth rate make investigations much easier. This thesis is concerned primarily with an understanding of the molecular events by which S. cerevisiae regulates one-carbon metabolism, especially that of the GDC at the level of gene expression, and which metabolites are key regulators of this control.
Chapter 3 examines how the transcription of genes encoding subunits of the GDC is controlled and determines control motifs in the promoter that mediate regulation of gene expression. It also examines whether the different subunits of the GDC are regulated
49 Introduction
coordinately. Mutants affected in known regulators such as Gcn4p, Baslp and nitrogen catabolic repression have been used to determine whether they also play a role in the overall regulation of the CCV genes.
Chapter 4 is concerned with the protein-DNA/protein-protein interaction studies to identify proteins which specifically interact with the "one-carbon" response control motif detailed in chapter 3. It also addresses which effector molecule signals the physiological status within cells, thus transferring nutritional information to achieve correct transcription of the necessary genes.
Since the genome of S. cerevisiae has been completely sequenced, it is possible to investigate genome-wide analysis of transcriptional control. Chapter 5 uses this approach to gain an insight into the overall transcriptional processes occurring in the cell when a single inducer of the one-carbon system is added to the cells to provide a foundation for establishing the extent of this regulatory system in yeast and to assist in identifying genes that encode as yet uncharacterised components of one-carbon metabolism.
50 Materials & Methods
Chapter 2: MATERIALS AND METHODS
2.1 Materials.
2.1.1 General materials and reagents.
Amino acids, folic acid, sodium tetrahydrofolate (THF) and folinic acid (5-
formyltetrahydrofolate), sulphanilamide, methotrexate, bromophenol blue, xylene cyanol,
ethidium bromide (EtBr), nitrophenyl-B-D-galactopyranoside (ONPG), acrylamide, bis
acrylamide, sodium dodecylsulfate (SDS), ethylenediaminetetraacetic acid (EDTA), 2-
mercaptoethanol, polyethylene glycol (4000), piperizinediethanesulfonic acid (PIPES),
N, N,N',N'-tetramethy lethy lenediamine (TEMED), pheny !me thy !sulfonyl fluoride
(PMSF), Ficoll (Type 400) and dithiothreitol (DTT) were obtained from Sigma-Aldrich
(NSW, Australia). ICN Biomedicals (NSW, Australia) and Progen (Qld, Australia)
supplied ampicillin, caesium chloride, agarose (DNA grade) and 5-bromo-4-chloro-3-
indoyl-B-D-galactopyranoside (X-gal).
Yeast extract, tryptone, bacteriological peptone, and agar type 1 and type 3 were
purchased from Oxoid Ltd. (NSW, Australia). Ammonium sulphate was purchased from
Sigma-Aldrich (NSW, Australia). Difco Laboratories (Detroit, USA) supplied yeast
nitrogen base (without amino acids and ammonium sulphate).
Bradford's protein reagent is distributed by Bio-Rad Laboratories (NSW,
Australia). Reinforced nitrocellulose (Hybond1 "-C) and Nylon (Hybond1 M-N+)
membranes were supplied by Amersham-Pharmacia (NSW, Australia).
All other materials and reagents were of high quality available and obtained from
various commercial vendors.
2.1.2 Radiochemicals and Autoradiograph. cx-[32P]dATP and cx-[32P]dCTP (3000 Ci/mmol) were purchased as aqueous solutions from Amersham-Pharmacia (NSW, Australia).
Polaroid instant black and white film Type 665 and Type 667 were purchased from Foto Riesel (NSW, Australia). Autoradiography diagnostic film (Kodak)
51 Materials & Methods
Biomax™-MR was purchased from Integrated Sciences (NSW, Australia). NEN™ Life
Science supplied Ret1ection™ Autoradiography Film NEF- (distributed by AMRAD,
VIC, Australia).
2.1.3 Enzymes and related materials.
Restriction and modifying enzymes, T4 DNA ligase, T4 polynucleotide kinase,
DNA polymerase I (Kienow fragment), pancreatic ribonuclease (RNaseA) and dNTPs were purchased from New England Biolabs (distributed by Genesearch Pty. Ltd., QLD,
Australia), Amersham-Pharmacia (NSW, Australia), or Boehringer Mannheim (NSW,
Australia). Taq polymerase was from Perkin-Elmer (NSW, Australia). Random primer labelling kits (MegaprimeTM) were purchased from Amersham-Pharmacia (NSW,
Australia). Crude Helix pomaria B-glucuronidase was supplied as a 125 U/Jll solution from ICN Biomedicals (NSW, Australia). Reverse transcriptase (SuperScriptTM,
RNaseH free) was from Life Technology (VIC, Australia).
2.1.4 Oligonucleotides.
Oligonucleotides (1995-1996) were synthesized on a Beckman 1000 DNA synthesizer, and purified with Beckman UltrafastTM cleavage and deprotection kits according to the manufacturer's specifications. After 1997, oligonucleotides were obtained from Pacific Oligos (QLD, Australia).
All nucleotides are shown in 5' to 3' orientation, and bases which alter the wild type sequences are shown in bold for those which were used as primers in site-directed mutagenesis. Numbers in parentheses indicate the binding site relative to the start codon ofGCV2.
GCV2-IR accgtagtaacccttacc (+427 to +444)
GCV2-2F ttgaggtatagaattctccttttcgg ( -282 to -257)
GCV2-3F cggagtcatgaattccagaggagtcat (-259 to -232)
GCV2-4F aggagtcatcgaattcagcattgagc (-241 to -223)
GCV2-5F gaaggaccttgagaattcagtcgaaagatc (-212 to -183)
52 Materials & Methods
The following five pairs of oligonucleotides were designed to anneal to give
EcoRI cohesive-ends so that could be inserted at the EcoRI site of pPCR2.
GRElT: aattgaggtata GREN!D: aatttatacctc
GRE2T: aatttcttgaggtata GREN2D: aatttatacctcaaga
GRE3T: aatttcttgaggt GREN3D: aattacctcaaga
GRE4T: aatttgactcttctt GREN4D: aattaagaagagtca
GREST: aattgaggtatagactcct GRENSD: aattaggagtctatacctc
The following oligonucleotides were made for site-directed mutagenesis (section
2.3.6) of either putative transcription factor binding sites or of the glycine-responsive region. Bold type indicates mutated nucleotides. Numbers in parentheses indicate the binding site relative to the start codon of GCV2.
MGCN4A catgttacccggttggtaccttacccgacatctc ( -325 to -292)
MGCN4B cccgacatctctggtacctcttgaggtatagac ( -282 to -257)
CTTl acatctctgactaggcttgaggtatagac (-298 to -269)
CTT2 tctctgactcttagggaggtatagactcc (-295 to -266)
Mcttkpn cccgacatctctgactggtaccgaggtatagactcctcc (-302 to -264)
GRR1 cgttacccgacatctctgactagtaccgaggtatagactc (-307 to -268)
GRR2 ggttgaatcgttacccgacatctctgactagtaccgagg (-315 to -277)
Primer 1(P1) acgaccgagcgcagcgagtc
Primer 2(P2) cgtacgtaatgcctgcaggtcgactctag
Primer 3(P3) gggagcaagcttgcatgcct
Primers 1, 2 and 3 were also used for site-directed mutagenesis (section 2.3.6) using PCR method and made based on Yip35(X)/Yip35(X)R. Bold letters indicated in primer 2 are mismatches for the purpose of site-directed mutagenesis (Mikaelian and
Sergeant, 1992). Primer 1 was also used for the sequencing of Yip35(X)/Yip35(X)R based constructs.
53 Materials & Methods
GCN4F caatctaccagccacacagctc
GCN4R cttctctccctgtcatactc
DBaslF cgaacaatgcgatgagccagacg
BASlTER gtgtacaaggcaaagttctagattatgcaaaatcgccg
BAS2PRO agacattgaagagagctcgacaagtcacgc
BAS2TER ccacctataacgcaagcttgtaaatatctatataccc
These oligonucleotides were used for amplification of the open reading frames of
GCN4, BASi, and BAS2 to confirm deletions (section 2.5.3).
310F gttacccggttgactcgagacccgacatctctg ( -325 to -290)
260R gtcatgactccgaactcgaggagtctatacc ( -278to -248)
TgrrF tcgacttgttcatcgccgtgacttctttcggcaggg (-193 to -162)
TgrrR tcgaccctgccgaaagaagtcacggcgatgaacaag ( -193 to -162)
The primers 310F and 260R were used to generate DNA fragment by PCR from
GCV2 genes for gel shift assays (section 2.8.2) and for cloning into the heterologous promoters. Bold indicates mutated sequences to create restriction sites and numbers in parentheses indicate the binding site relative to the start codon of GCV2. The GCV1 fragment -193 to -162 with Xhol cohesive ends was produced by annealing oligonucleotides TgrrF and TgrrR after phosphorylation (section 2.3.3), it was used for gel shift assays and cloning into the heterologous promoters.
2.1.5 DNA and vectors.
Yip35(X)/Yip35(X)R: Integrative S. cerevisiae/E. coli shuttle vectors suitable for construction of yeast promoter fusions. Ylp356, Ylp357, and Ylp358 have the multiple cloning site (MCS) juxtaposed to maintain the three possible open reading frames, and they contain the URA3 gene for selection in yeast (Myers eta/., 1986). Ylp35(X)R vectors are identical to Ylp35(X) except for an inverted MCS.
pTZlS/19: Multifunctional phagemid vectors based on pUC18/19 (Phannacia; obtained from D. Sinclair, UNSW).
54 Materials & Methods
pLGD.-312S and pLGD.-312SS: The pLGD.-312S is an episomal plasmid that contains the promoter of the CYC I gene fused to the /acZ repatter gene (Guarente and
Hoar, 1984). This promoter contains intact upstream activation sequences (UAS) and a
Xhol site between the UAS and the TATA box, which was used for introducing test sequences. The plasmid pLGD.-312SS is identical to pLGD.-312S except that the UAS sites in the promoter region of the CYCJ gene were removed by cutting with Srnal and Sail, then the plasmid was religated after filling in the Sail site.
The CCVI gene cloned in YEp 13 was obtained from McNeil eta/. ( 1997). This plasmid contains Hindiii fragments (5.5-6 kb) of the CCV1 gene with most of the upstream (at least I kb) and coding regions of the CCVI gene, but lacking the sequence encoding the C-terminal region.
pB 1559: This YipS-based plasmid with the BASI gene deleted for the Spei
Xhoi fragment ( -179 to 520 bp relative to start codon) was used for chromosomal deletion (section 2.5.3) of the BASI gene (Arndt eta/., 1987).
2.1.6 Bacterial and S. cerevisiae strains.
Escherischia coli strains
JM101: F' traD36 proAB lac[q L1(/acZ) Ml5 proA +B+ /supE thi L1(/ac-proAB)
(Messing, 1979).
JM109: F' traD36lacJq L1(lacZ) MIS proA+B+ /el4(McrA-) L1(lac-proAB) thi gyrA96
(Nal') endAJ hsdR17 (rk- mk+) re/Al sup£44 recAI (Yanisch-Perron, et al., 1985)
NM522: F' lac/4 L1(lacZ) M15 proA+B+ /supE thi L1(lac-proAB) L1(hsdMS-rncrB)5(rk-
(Gough and Murray, 1983)
Saccharomyces cerevisiae strains:
BWG1-7A (Y3): MATa adel-100 his4-519/eu2-3leu2-112 ura3-52
(Guarente and Mason, 1983)
Fll3: MATa inol can] ura3-52
F212: MATa inol canl ura3-52 gcn4-103
55 Materials & Methods
F212 is isogenic to F 113 except for deletion of the GCN4 gene. These strains were provided by Dr. A. G. Hinnebusch and obtained from Dr. C. Grant (UNSW,
Australia).
23344c: MAT a ura3 (M. Grensen, unpublished)
30078c: MATa ura3 uga43L1 (Coornaert eta!., 1992)
30505b: MATa ura3 gln3L1 (Andre eta!., 1995)
30495a: MATa ura3 uga43L1 gln3L1 (Andre eta!., 1995)
SBSIO: MATa ura3 gzf3L1::LEU2leu2 (Soussi-Boudekou eta!., 1997)
SBS21: MATa ura3 nillL1::KanMX2 (Soussi-Boudekou eta!., 1997)
50000b: MATa ura3 gzJ3L1::LEU2 uga43L1 (Soussi-Boudekou eta/., 1997)
5002ld: MATa ura3 gzj3L1::LEU2 nillL1::KanMX2 (Soussi-Boudekou et al., 1997)
50027c: MATa um3leu2 gln3L1 nillL1::KanMX2 (Soussi-Boudekou et al., 1997)
27033d: MATa ura3 leu2 (J.-Y. Springael, unpublished)
These nitrogen-regulatory mutants were donated by Dr. B. Andre (Universite
Libre de Bruxelles, Belgium).
Y475: MATa/a leu2-3,112 ura3-52 trpl-289 his3-deltal, FOLl/foll::KanMX
(J. H. Hegemann, unpublished; obtained with permission from M.
Breitenbach, Austria)
2.1.7 Media.
E. coli maintenance and growth media
E. coli cultures were maintained on minimal medium containing: 10.5 gil
KzHP04; 4.5 g/1 KH2P04; 1.0 g/1 (NH4)zS04; 0.5 g/1 tri-sodium citrate; 0.2 g/1 MgS04;
2.0 g/1 D-glucose; 0.5 ml/1 thiamine-HCI (5 mg/ml) and 16 g/1 agar type 3 (technical grade). Colonies were cycled between minimal and growth media every 2 months to maintain the bacterial F' episome. E. coli was grown on YT media consisting of 8 g/1 tryptone, 5 g/1 yeast extract and 5 g/1 NaCI. For plasmid extraction, or for the preparation of competent cells, 2x YT medium was used which consisted of 16 g/1 tryptone, 10 g/1
56 Materials & Methods
yeast extract, 5 g/1 NaCl and 5 ml/1 glycerol. Antibiotic selective media contained 50
Jlg/ml ampicillin. B-galactosidase selective plates contained 50 Ill of X-gal (20 mg/ml in
dimethylformamide) spread evenly over the surface of the plate.
Saccharomyces cerevisiae media
Unless specified otherwise, S. cerevisiae was grown in YEPD medium which is
comprised of YEP medium (20 g/1 bacteriological peptone and 10 g/1 yeast extract) with
the addition of 20 g/1 D-glucose. YEPG and YEPL medium consisted of YEP medium
with the addition of 20 ml/1 glycerol or lactate respectively. Solid medium contained an
additional 20 g/1 agar. S. cerevisiae was maintained on solid YEPD medium.
Glucose minimal medium (Dmin) consisted of 20 g/1 D-glucose, 1.7 g/1 Difco
yeast nitrogen base without amino acids and ammonium sulphate (YNB) and 5 gil
ammonium sulphate. Dmin+gly and Dmin+ser medium consisted of Dmin medium with
10 mM glycine or L-serine respectively. Minimal medium with an amino acid as the sole
nitrogen source (GL Ymin, GLNmin, ARGmin, ASNmin and PROmin) contained 20 g/1
glucose, 1.7 g/1 YNB and 15 g/1 of respective amino acid (glycine, L-glutamine, L arginine, L-asparagine and L-proline). Other amino acids for the auxotrophic requirements were added to the minimal media at a concentration of 40 mg/1.
Yeast transformants were selected and maintained on 'drop out' media consisting of 20 g/1 D-glucose, I. 7 g/1 YNB and 5 g/1 ammonium sulphate, 5 g/1 agar, 40 mg/1 of each auxotrophic requirement, except for the amino acid used for selection of the trans formant.
Sporulation medium consisted of 20 g/1 potassium acetate, 20 g/1 yeast extract, and 0.5 g/1 D-glucose.
2.2 General Procedures
2.2.1 Sterilization and containment of biological material.
Plastic and glass items and heat-stable solutions were sterilized by autoclaving at
120oC (125 kPa) for 15 min. Heat-labile solutions were sterilized by passing through a
0.2 ~-tm sterile membrane filter.
57 Materials & Methods
Glassware and metallic objects for handling RNA were sterilized by dry heat in an
180oC oven overnight. Solutions for RNA work were treated with DEPC for a minimum of 5 h and autoclaved.
All biologically contaminated material was autoclaved at 125 kPa for 20 min prior to disposal.
2.2.2 Buffers and stock solutions.
TE buffer (pH 7.4): standard buffer for storage of DNA; 10 mM Tris-HCI (pH
7.4) and 1 mM EDT A (pH 8.0).
T AE buffer: buffer used for agarose gel electrophoresis; 40 mM Tris-acetate, 1 mM EDT A (pH 8.0).
TBE buffer: buffer used for polyacrylamide/agarose gel electrophoresis; 45 mM
Tris-borate, and I mM EDT A (pH 8.0).
30% Acrylamide: used for polyacrylamide gel for separation and isolation of
DNA; dissolve acrylamide (29% w/v) and bis-acrylamide (1% w/v) in water, filtrate and store the solution in a dark bottle at room temperature.
10% Acrylamide: used for electrophoretic mobility shift assay; dissolve acrylamide and bis-acrylamide in 98.8:1.2 ratio in water to make up 10% stock solution, filtrate and store the solution in a dark bottle at room temperature.
40% Acrylamide: used for sequencing gel; dissolve acrylamide (38% w/v) and bis-acrylamide (2% w/v) in water, filtrate and store the solution in a dark bottle at room temperature.
PMSF: stock solution (10-100 mM) was prepared by dissolving PMSF in isopropanol and stored at -20°C.
3 M Sodium acetate (pH 5.2): used for ethanol precipitation; prepared by dissolving sodium acetate in water and adjusting the pH with glacial acetic acid.
Single-stranded (carrier) DNA: used for yeast transformations and Southern hybridisations; prepared by dissolving salmon sperm DNA (10 mg/ml) in water and the
DNA was sheared by passing it rapidly through a 17-gauge needle at least ten times
58 Materials & Methods before being boiled for 10 min and rapidly cooled on ice. The stock solution was stored at -20'C in small aliquots.
20x SSC: used in Southern analysis and gene array analysis; prepared as 3 M
NaCl, 300 mM tri-sodium citrate.
6x gel-loading dye: 0.25% bromophenol blue, 0.25% xylene cyanol, 15% (v/v)
Ficoll (Type400) in water and stored at room temperature.
Sequencing gel-loading dye: 95% (v/v) formamide, 10 mM EDTA, I% bromophenol blue, 1% xylene cyanol.
Footprint buffer: buffer for DNA-protein binding reaction; 0.2 M Tris-HCl (pH
8.0), 0.7 M KCl, 50 mM MgCl2, 5 mM CaCl2, 5 mM DTT, 1 mM EDTA and 70% (v/v) glycerol.
2.2.3 Substrate and enzyme stock solutions.
X-gal: stock solution was prepared as 2% (w/v) in N,N-dimethyl formamide and stored at -20'C in an aluminium foil-wrapped tube.
Z-buffer: buffer used for 13-galactosidase assay; 16.1 g/1 Na2P04 7 H20, 5.5 g/1
NaH2P04.H20, 0.75 g/1 KCl, 0.246 g/1 MgS04 7H20, 2.7 ml/1 2-mercaptoethanol and adjusted to pH 7.0
ATP (10 mM) was dissolved in 10 mM Tris-HCl (pH 7.0) and the pH was adjusted with 0.1 M NaOH to pH 7.0.
Proteinase K: stock solution was prepared by dissolving proteinase K in water
(20 mg/ml), and stored at -20'C. For the reaction, it was incubated at 3TC in reaction buffer containing 10 mM Tris-HCl (pH 8.0), 5 mM EDT A, 0.5% SDS at a concentration of 50 ~-tg/ml.
Pancreatic RNase (RNaseA) was dissolved in 10 mM Tris-HCl (pH 7.5) at a concentration of 10 mg/ml and boiled for 10 min to remove any contaminating DNases, then stored in aliquots at -20'C.
13-glucuronidase: cell wall digestion enzyme; prepared as a 12.5 U/~-tl solution in
100 mM sodium citrate/potassium phosphate buffer (pH 5.3). Debris was removed by centrifugation ( 12 000 x gin a microcentrifuge for I min) before use.
59 Materials & Methods
Sodium tetrahydrofolate stock solution (0.07 M) was prepared by adding
tetrahydrofolic acid to I M B-mercaptoethanol, and adjusting the pH to 7 with 2 N NaOH.
To avoid rapid oxidation of tetrahydrofolate to dihydrofolate in the presence of
atmospheric oxygen, solutions were bubbled with argon for I min and the tubes were
kept purged. Stocks were stored frozen in sealed ampules at -70'C in 20 111 aliquots.
2.2.4 Preparation of dialysis tubing.
Dialysis tubing was cut to the desired length (15 em) and passed through
successive washes of distilled water, NaHC03 (SO'C), 2 I of 10 mM Na2EDT A (pH 7)
for 3 min, and finally distilled water (SO'C) for 30 min. Tubing was stored at 4'C in a
30-40% (v/v) ethanol solution. Before use, tubing was either washed in water or
squeezed to remove excess ethanol.
2.3 DNA Manipulations
2.3.1 Restriction digestion of DNA.
Restriction digest was performed according to the manufacturers' specified
conditions, with regards to dilution of enzyme, buffers and temperature. Complete
digestion typically required l-3 U of enzyme per 11g DNA incubated for 2-15 h.
Restriction enzymes were removed by either electrophoresis, denaturation at 70'C for 15
min, or phenol extraction (section 2.4.1) before subsequent enzymatic reactions.
Digestion of total genomic DNA for Southern analysis generally required 10 U of enzyme per !lg DNA and was performed overnight.
2.3.2 Blunt end generation.
For 5'-overhanging ends, the 5' to 3' DNA polymerase activity of Klenow fragment was used for fill-in reaction of 3'-recessed ends. DNA was dissolved (50 ng/!11) in the reaction mixture containing reaction buffer, 33 11M dNTP's and Klenow fragment ( l U per !lg of DNA) and the reaction was carried out at 25'C for 15 min. The reaction was stopped by adding EDT A to a final concentration of 10 mM and heating at
60 Materials &Methods
75'C for 10 min. After inactivation, ethanol precipitation was performed to remove excess EDTA that may interfere with subsequent reactions.
For 3'-overhanging ends, 3' to 5' exonuclease activity of T4 DNA polymerase was used. DNA was dissolved (50 ng/fll) in the reaction mixture containing reaction buffer, 100 fJ.M of each dNTP's and T4 DNA polymerase (1 U per fJ.g of DNA) and the reaction was carried out at 12'C for 20 min. The reaction was stopped by heat inactivation (75'C for 10 min).
2.3.3 Phosphorylation of synthetic oligonucleotides
T4 polynucleotide kinase catalyzes the transfer of the terminal phosphate of ATP to the 5'-hydroxyl termini of DNA or synthetic nucleotides. To the tube containing 1 to
10 fJ.g of oligonucleotides, were added to the final concentrations, 70 mM Tris-HCl (pH
7.6), 10 mM MgCl2, 5 mM OTT, I mM ATP, 50 fJ.g/ml BSA and 20 U of T4 polynucleotide kinase. The reaction was carried out at 3TC for 1 h, and stopped by adding I fll of 0. 5 M EDT A.
2.3.4 Insertion of small DNA fragment.
Small DNA fragment (8-20 bp) insertion into a restriction site of vector sequences was achieved by first designing a pair of oligonucleotides which would anneal to have overhanging ends of a restriction enzyme site. For insertion of multiple DNA fragments, the 5'-end of synthetic oligonucleotides must be phosphorylated (section 2.3.3.) before annealing. Annealing was performed by boiling a solution containing equimolar amounts of a pair of oligonucleotides in an Eppendorf tube and slow cooling overnight in I I of water. After annealing, the product was used for ligations with a vector that had been restriction digested to have compatible ends with the annealed products. Ligation with small DNA fragments was carried out at 10'C overnight.
2.3.5 Ligation of DNA.
Vector and inselt DNA were combined in molar ratios ranging from 1:2 to 1:10 in a total reaction volume of 20-40 fJ.l. Ligation reactions were performed with T4 DNA ligase (0.5 U) in ligase buffer consisting of 50 mM Tris-HCI pH 7.5, 5% (w/v) PEG
61 Materials & Methods
4000, 10 mM MgCI2, I mM DTT and I mM ATP. Reactions were incubated at 16'C overnight. For blunt-end ligation, 5 U of ligase was used per reaction.
2.3.6 Site-directed mutagenesis.
Site-directed mutagenesis was performed as described in Mikaelian and Sergeant
(1992). The oligonucleotides used for site-directed mutagenesis are listed in section
2.1.4. Base changes were chosen to accommodate appropriate restriction enzyme sites or/and to abolish putative protein binding sites. Polymerase chain reactions (PCR) were carried out to amplify DNA fragments using the oligonucleotides. The PCR was carried out for 30 cycles (94'C, I min; 48'C, 2 min; 72'C, 2 min). The resulting DNA fragments were purified with PCR® Spinclean"1 DNA purification kit (Progen) according to the manufacturer's specification and were then cut with appropriate restriction enzymes for cloning into vector.
2.4 Preparation of DNA and RNA.
2.4.1 Plasmid DNA preparation and purification
Small scale plasmid preparation was performed as in Ish-Horowitz and
Burke (1981) with modifications. For plasmid preparations, E. coli was grown to stationary phase as a patch(~ 6 cm2) on a YT-ampicillin plate or grown in 2 ml of YT ampicillin medium. Cells were scraped off with a toothpick (plate) or collected by centrifugation (medium), and suspended in 100 fll of 50 mM glucose, 25 mM Tris-HCI,
10 mM EDTA at pH 8.0. A 200 fll freshly combined solution of I% SDS and 0.2 M
NaOH was added to the cells and mixed. After 10 min on ice, 150 fll of ice-cold potassium acetate (pH 4.8) was added and vortexed briefly. After incubating on ice for 5 min, the solution was centrifuged in a microcentrifuge tube for 5 min. The supernatant was then phenol extracted and the DNA was ethanol-precipitated as described below in this section and resuspended in 20-50 fll of TE buffer (pH 7 .5) with 1 fll of 10 mg/ml
RNase.
62 Materials & Methods
For a larger quantity of plasmid DNA, 250 ml culture was grown to stationary phase overnight in 2xYT plus ampicillin. DNA was extracted by the method described above, with the volumes scaled accordingly. DNA was purified by sedimentation on a
CsCI density gradient as described below.
Midi-scale preparation was used routinely for Midi (up to 20 IJ.g) preparation of plasmid using QIAGENT" Plasmid Mini Kit. Cells were grown in 25 ml YT medium and the DNA was prepared according to the manufacturer's protocol.
Polyethylene glycol (PEG) precipitation was used for purification of plasmid DNA for sequencing. The method was based on Sambrook et al. (1989).
Plasmid DNA was isolated by large scale preparation (250 ml culture) and PEG precipitation was carried out with 1 vol of 13% (w/v) PEG 4000: 2M NaCI. After thorough vortexing, the sample was left overnight at 4'C. After centrifugation at 13,000 x g for 30 min at 4 'C, the supernatant was discarded and the DNA pellet was washed with 70% (v/v) ethanol. The ethanol solution was removed after being centrifuged and the DNA was dried either in air or under vacuum.
CsCI density gradient method was employed to purify plasmid DNA during large scale preparation. To partially purified DNA samples was added 80 IJ.I EtBr (l 0 mg/ml), 3.15 g CsCI and the total volume was brought to 3.5 ml resulting in a density of
!.59- 1.60 g/ml. This sample was centrifuged at 85 000- 90 000 x g for at least 6 h, at
20'C. The EtBr-dyed DNA was collected and extracted several times with an equal volume of CsCI-saturated isopropanol to remove EtBr, followed by dialysing two times against TE buffer (2 I) for 3 h at 4 'C.
Phenol extraction was performed by the addition of one volume of 1: I chloroform/phenol. The phenol was equilibrated with TE buffer (l 0 mM Tris-HCI, 1 mM EDT A, pH 8.0) before use. Samples were then vortexed thoroughly and centrifuged in a microfuge for 1 min at 12,000 x g. The upper aqueous phase was collected and 1 vol of phenol/chloroform (I: I) extracted and the extraction can be repeated to improve the
63 Materials & Methods purity of DNA. Extraction with I vol of chloroform was used to remove residual phenol in DNA solution.
Ethanol precipitation was carried out to precipitate DNA by adding 0.1 vol of
3 M sodium acetate (pH 5.2) and 2.5 vol of absolute ethanol. The efficiency with which very short DNA fragments (less than 100 bp) were precipitated can be improved by adding additional MgC1 2 to a final concentration of 10 mM. After mixing briefly, DNA was precipitated at -70'C for a minimum of 15 min, then pelleted in a microcentrifuge for
15 min at 12,000 x g. The ethanol solution was removed and washed with I vol of cold
70% (v/v) ethanol. The DNA was pelleted in a microfuge for 5 min, the ethanol solution removed, and the DNA was dried either in air or under vacuum.
2.4.2 Yeast genomic DNA preparation
Yeast genomic DNA was isolated by the method of Hoffman and Winston ( 1987) for rapid genomic DNA preparation from Saccharomyces cerevisiae. 10 ml of culture grown to stationary phase yielded about 20 !lg of DNA, of which 2-4 !lg was used for
Southern hybridisation. DNA was stored at -20'C in TE buffer (pH 7.4).
2.4.3 RNA preparation
Yeast total RNA preparation Yeast total RNA was isolated using the method of Schmitt et al. (1990). Generally, a 50 ml culture was grown to an OD6oo of 0.5 and harvested by centrifugation. The pellet was resuspended in 400 111 of AE buffer (50 mM sodium acetate, pH 5.3; 10 mM EDTA), 40 111 SDS (10% w/v) and 400 111 phenol
(equilibrated with AE buffer) and vortexed for I min, followed by incubation at 65'C for
4-5 min and then quickly chilled in dry ice/ethanol until phenol crystals appeared. The aqueous phase was collected in a microfuge for 2 min and extracted with phenol/chloroform (section 2.4.1). The phenol/chloroform extraction was repeated for higher purity RNA. The RNA was precipitated with the addition of 40 111 of 3M sodium acetate (pH 5.2) and 2.5 vol ethanol. RNA was resuspended in either TE buffer/0.1%
SDS solution or TE buffer with 5 mM DTT and I U/!11 RNAsin.
64 Materials & Methods
mRNA preparation Dynabeadsr" Oligo (dTl2s kit (Dynal Pty. Ltd., Australia) was used for poly A RNA (mRNA) isolation from total RNA. Procedures were followed according to the manufacturer's specification. 75 )lg of total RNA yielded up to 5-7 )lg of mRNA. Prepared mRNA was stored in -70'C for up to 6 months.
2.4.4 Estimation of DNA and RNA concentration and purity.
Concentrations of DNA and RNA were estimated by the absorbance of an appropriately diluted sample at 260 nm. An absorbance of 1.0, through a 1 em light path corresponds to a dsDNA concentration of approximately 50 )lg/ml, and RNA concentration of 40 )lg/ml. The purity of nucleic acids was estimated by determining the
A26olA2so ratio which was 1.8-2.0 for pure solutions.
2.4.5 Sequencing
Sequencing was performed with the Applied Biosystems Model 373A Automated
DNA Sequencing System (UNSW, Australia). ABI PRISMT" BigDyerM Terminator was used for PCR-based dye-terminating sequencing. PCR for the dye termination reaction was carried out in a volume of 20 )ll for 25 cycles (96'C, 30 sec; 50'C, 15 sec;
60'C, 4 min). Products were ethanol precipitated and submitted to the DNA sequencing unit (UNSW, Australia).
2.5 Cell manipulations.
2.5.1 General yeast genetic techniques
Diploids were sporulated on acetate minimal sporulation plates (section 2.1.7) for
4-5 d. Before dissection, tetrads were incubated at room temperature in 50 )ll B glucuronidase stock solution (section 2.2.3) for 20-30 min. After transferring tetrads on to YEPD plates (made with agar type No.I), spores were separated with a Singer micromanipulator (Singer MSM system"').
2.5.2 Transformations and transfections.
65 Materials & Methods
E. coli transformation. Competent cells were prepared by calcium chloride treatment as described by Messing (1983). To ISO 111 of competent cells was added 20 ng of the plasmid, or the ligation reaction product and the mixture were incubated on ice for 15-20 min, heat-shocked at 42'C for 2 min. To the transformed cells I ml of YT medium was added and incubated for I h at 3TC to allow expression of the antibiotic resistance gene. The cells were spun down for 10 sin a microfuge. After pouring off the supernatant, the cells were resuspended in the residual medium. The suspension was plated on to selective plates and incubated at 3TC overnight.
S. cerevisiae transformation was done as described in the procedures of
Geitz et a/. (1992). Yeast were grown to an OD6oo of 0.8 - 1.1 in YEPD or, alternatively, overnight culture in 10 ml tube was diluted 10 times in YEPD and further grown for 4-5 h before transformation. Cells were harvested by centrifugation at 5,000 x g for 5 min. The cells were then washed twice in lithium acetate/TE buffer (I 00 mM lithium acetate, pH 7.5; 10 mM Tris buffer, pH 7.5; I mM EDTA, pH 8.0). To 50 ~-tl aliquots of cells was added I ~-tl carrier ssDNA (section 2.2.2) and DNA (5 11g for integrative vectors and I ~-tg for episomal vectors) and a mixture of 240 ~-tl polyethylene glycol4000, 30 ~-tl !Ox TE buffer and 30 111 I M lithium acetate. The mixture was mixed well and incubated for 30 min at 30"C with shaking. The cells were then heat-shocked for 15 min at 42'C before plated on selective media for 3-4 d.
2.5.3 Yeast deletion mutant generation.
The pop-in/pop-out gene replacement technique was used for yeast gene deletion mutant generation as described in Rothstein (1991) using mutant selection on minimal media containing 5-fluoro-orotic acid (I g/1). Integration of a plasmid containing the
URA3 gene and partly-deleted target gene by homologous recombination results in a tandem repeat of the gene (one mutant and one wild-type copy) carried on the plasmid flanking the plasmid sequences and the U RA3 gene. Homologous recombination between the repeated segments will leave the mutant or wild-type alleles behind and these pop-out strains can be selected on plates containing 5-fluoro-orotic acid. The deletion mutant was confirmed by PCR using primers specific to the target gene.
66 Materials & Methods
2.5.4 Yeast petite mutant generation
To make petite mutants of S. cerevisiae, cells were streaked and grown on a
YEPD plate (section 2.1.7) containing 20 j.!g/ml ethidium bromide. After cells were grown for 3-4 d, growth of the cells was tested on YEPG and YEPD. Petites cannot grow on YEPG since they can not use glycerol as a carbon source.
2.6 Gel Electrophoresis
2.6.1 Agarose gel electrophoresis.
Agarose gel electrophoresis was used for the visualization and isolation of DNA fragments greater than 500 bp. DNA samples were loaded with gel loading-dye (section
2.2.2) Horizontal slab gels (0.5% - 2.0%) were run in T AE or TBE buffer (section
2.2.2) and electrophoresed at 40-60 rnA. DNA was visualized on an ultra-violet transilluminator. A Polaroid Mamiya RB-67 camera with Polaroid type 665 instant black and white film was used to photograph EtBr-stained DNA in gels under UV illumination
(254 urn) exposed for 15 s.
For the isolation of DNA fragments (0.5 - 10 kb), excised gel fragments were centrifuged through 4 mm of packed siliconised glass wool in an Eppendorf microcentrifuge at 1,000 x g for 10 min. Alternatively, DNA fragments were isolated from gel with BRESAspin™ Gel Extraction Kit (Bresatec, Pty. Ltd., SA, Australia).
For the separation and extraction of small fragments of DNA (smaller than 500 bp), polyacrylamide gels were employed.
2.6.2 Polyacrylamide gel electrophoresis.
For the visualization and isolation of DNA fragments smaller than 500 bp, 8 -
15% (w/v) polyacrylamide gels were prepared from 30% acrylamide/bis-acrylamide
(29: I) stock solution and 5x TBE buffer. Polymerization of the gel was achieved by the addition of 180 j.!l of 10% (w/v) ammonium persulphate and l8j.!l TEMED per 20 ml of gel mixture. After pre-electrophoresis of the gel for 20 min in TBE buffer, DNA samples were loaded with 6x gel-loading dye (section 2.2.2) and electrophoresed at 8 V cm·1.
67 Materials & Methods
DNA bands were visualized on UV transilluminator by soaking the gel in 0.5 IJ.g/ml ethidium bromide in TBE buffer for 30-45 min.
For the isolation of small DNA fragments(< 500 bp), DNA bands were excised from the gel and eluted overnight in the elution buffer containing 0.5 M ammonium acetate, 10 mM magnesium acetate, I mM EDTA and 0.1% SDS at 55"C. DNA was then ethanol precipitated, dried and redissolved in TE buffer or water.
Sequencing gels for footprinting were prepared in TBE buffer containing 50%
(w/v) urea and 8% (w/v) acrylamide/bis-acrylamide (19:1). A solution ofBind-SilaneTM
(13 IJ.l), 130 IJ.! acetic acid (10% v/v) and 5 ml ethanol was applied to the glass backing plate to promote its adhesion to the gel. Polymerization was achieved by the addition of
350 f.ll of 10% (w/v) ammonium persulphate and 60 f.!l TEMED per 50 ml of gel mixture.
Gels were pre-electrophoresed for 20 min at 1800 V and run at 2500 V at 55'C. After washing in 10% (v/v) methanol: 10% (v/v) acetic acid for I h the gels with agitation, gels were air dried on the backing plate and autoradiographed (section 2.7.5) for 1-2 d.
2.7 Radioactive Labelling and Autoradiography.
2. 7.1 End labelling
End labelling of DNA was carried out with E. coli DNA polymerase I (Klenow fragment) as described in section 2.3.2, except for the substitution of dATP or dCTP with 5 111 of [32P]a-dATP or 5 f.ll of [32P]a-dCTP per reaction.
2.7.2 Random hexamer labelling.
A random hexamer labelling kit (MegaprimeT") was used to label DNA. DNA
(50-100 ng) in 25 IJ.l of H20 was denatured at IOO'C for 5 min and then cooled rapidly on ice. After spinning down in a microcentrifuge for 10 s, 5 IJ.l of random hexamer solution, 5 f.!l of (lOx) reaction buffer, 41J.l of dGTP and dTTP, 41J.l of [32P]a-dATP (or dATP), 4 f.!l of [32P]a-dCTP (or dCTP), and 1-3 U of DNA polymerase (Klenow fragment) was added and incubated at 3TC for 15 min to 1 h. The extent of labelling was confirmed with 0.5 f.!l of reaction mixture by thin layer polyethylene imine (PEl)
68 Materials & Methods chromatography using 0.65 M sodium phosphate buffer (pH 6.3) and exposure to X-ray film for 5 min. The labelled probes were denatured for 5 min at lOO'C and chilled on ice before hybridization (2. 7.4 ).
2.7.3 eDNA labelling
eDNA labelling from mRNA was performed using reverse transcriptase. To 8 111 of mRNA (I !lg) solution, 2 111 of oligo(dT)J2-18 primer (0.5 !lgl!ll) was added and heated at 70'C for 10 min to remove secondary structure in the RNA. 2-3 11g of
Oligo(dT)25 was also added to the solution to eliminate the "poly A effect" (Jordan,
1998). After chilled on ice, to the tube was added 1111 of DTT (0.1 M), 1.5 111 of dNTP solution (10 mM each dATP, dGTP and dTTP), 10 111 of [32P]a-dCTP, 1.5 111 (300 U) of SuperScriptII RNaseH· Reverse Transcriptase (Life Technology) and 6 111 of reaction buffer (5x) containing 250 mM Tris-HCl (pH 8.3), 375 mM KCl and 15 mM MgCl2, and incubated at 3TC for 90 min. To remove unincorporated dNTPs, the probe was purified by a passage through a Nick Column TM (Sephadex® 050 column). The labelled probes are denatured for 5 min at lOO'C and chilled on ice before hybridization (2.7.6).
2. 7.4 Southern analysis
Digested genomic DNA (section 2.3.1) was separated on a 0.7% agarose gel which was rinsed with distilled water and DNA fragments were denatured with a 0.5 M
NaOH, 1.5 M NaCl solution for 30-45 min, and neutralized with a 1M ammonium acetate, 0.02 M N aOH solution for 30-45 min. After unidirectional transfer overnight
(Sambrook et al., 1989), the DNA was fixed on to the membrane by baking at 80'C for
1-2 h. A random hexamer labelled probe (section 2.7.2) was hybridised to the membrane in hybridization buffer (Rapid-Hyb™, Amersham) and washed as described by
Sambrook et al. ( 1989). The membrane was autoradiographed as described in section
2.7.5.
2.7.5 Autoradiography and Photography.
Autoradiography of sequencing gels was performed using BiomaxT''-MR. Other autoradiographs used Reflection TM Autoradiography Film NEF-. Exposure was
69 Materials & Methods generally between 12 and 36 h. For 32p exposures, intensifying screens were placed on both sides of the film which was exposed at -70"C. Alternatively, radioactivity was scanned by a Phosphoimager (Bio-Rad).
2. 7.6 Gene-array analysis
GeneFiltersTM (Research Genetics, Inc.) containing a total of 6144 gene open reading frames were hybridized with the labelled eDNA (section 2.7.3) after at least 2 h prehybridization in hybridization solution (MicroHyb™, Research Genetics, Inc.) at
42'C. To avoid the "polyA effect" (Jordan, 1998), oligo(dT)so (2 ~g per 1 ml of hybridization solution) was added in the prehybridization step. Hybridization was carried out for 2 d at 42'C. After hybridization, filters were washed in 2x SSC, I% SDS for 20 min at room temperature, and twice in 0.2x SSC, 0.1% SDS at 50"C for 1 h with gentle horizontal shaking. Autoradiography was performed with a Phosphoimager (Bio-Rad) with exposure for 1-2 d. To prevent the filters from drying out during exposure, they were placed on a piece of wet Watmann paper and wrapped with plastic wrap. To strip the filters after use, 500 ml of 0.5% SDS was heated 7-10 min in a microwave and poured into a box containing the filters and agitated for 1-2 h. The efficiency of stripping was monitored with a Geiger counter. Filters were stored humid between two sheets of wet Whatman paper wrapped with plastic wrap at 4 'C.
2.8 Protein-DNA Interaction study
2.8.1 Yeast protein extract preparation
Nuclear protein extraction
Nuclear protein extraction of Saccharomyces ce revisiae was prepared by the method of Stanway et al. ( 1987) with modifications. Yeast strain BWG 1-7 A was grown in 11 of an appropriate medium to an OD6oo of 1.0 at 30 oc. Cells were harvested (5,000 x g, 5 min), washed once with I M sorbitol, resuspended in 20 ml of 1 M sorbitol containing 2% glusulase and incubated with gentle shaking at 30 °C for 1 h. Protoplasts
70 Materials & Methods were harvested by centrifugation (3,000 x g, 5 min), washed in SPC buffer (I M sorbitol, 20 mM PIPES, 0.1 mM CaCI2, pH 6.4) and resuspended in 0.25 ml of the same buffer. 10 ml of a solution containing 9% (w/v) Ficoll, 20 mM PIPES, 0.1 mM
CaCI2, and I mM PMSF, pH 6.4 was added to the protoplasts. Nuclei were pelleted by centrifugation at 20,000 x g for 20 min at 4 °C and resuspended in 2 ml of SPC buffer containing I mM PMSF. After repelleting the nuclei were resuspended in 0.5 ml of a solution containing 0.6 M NaCI, 5 mM EDTA, 10 mM 2-mercaptoethanol, 10 mM Tris
HCI, pH 7 .5, 0.5 mM PMSF and left on ice for 30 min with occasional vortex mixing.
The nuclear protein extract was obtained after 30 min centrifugation at 13,000 x g (4 °C), and subsequently dialysed against buffer containing 10 mM Tris-HCI; pH 7.5, I mM
EDT A, I 0 mM 2-mercaptoethanol. Aliquots were stored at -70 C with final glycerol concentration of 15 % (v/v).
Protein preparation by heparin-Sepharose chromatography
Procedures were based on Ruet eta!. ( 1984) with modifications. Protein extracts were prepared with yeast cells grown in minimal media (Dmin) to an OD60o of 1.0 at 30
C. Cells were harvested by centrifugation (4,000 x g for 5 min at 4 C) and resuspended in breakage buffer (20 mM Tris-HCI pH 8.0, 1 mM EDTA, 10 mM B-mercaptoethanol,
10 mM MgCI2, 2 mM ZnS04, 1 mM PMSF, 0.3 M (NH4hS04, I ~-Lglml aprotinin, 0.5
~-Lglmlleupeptin, 0.7 ~-Lglml pepstatin A, 10% v/v glycerol) then were broken by agitation with acid-washed glass beads in a homogeniser using three treatments for 40s, with 5 min cooling on ice between each burst. After removing glass beads by centrifugation
(2,000 x g for 2 min), soluble proteins were collected by centrifugation (I 00,000 x g for
1 hat 4oC) and diluted with an equal volume of column buffer (20 mM Tri-HCl pH 8.0,
I mM EDTA, 10 mM 2-mercaptoethanol). The diluted sample was then loaded onto a heparin-Sepharose column previously equilibrated with column buffer at 4°C at a flow rate of I ml/min. The column was washed with column buffer containing 0.1 M
(NH4hS04 until unbound proteins had eluted and bound proteins were eluted with a linear gradient of column buffer containing (NH4l2S04 from 0.1 M to IM. Fractions were collected and dialysed against storage buffer (20 mM Tri-HCl pH 8.0, 50 mM KCl,
71 Materials & Methods
1 mM DTT, 0.2 mM EDT A and 10% v/v glycerol) at 4 °C and aliquot of each fraction were stored at -70°C.
2.8.2 Electrophoretic Mobility Shift Assay (EMSA)
The 5'-overhanging ends of DNA fragment to be assayed were labelled with the
Klenow fragment of DNA polymerase in the presence of [o:-32P]dATP/dCTP and the labelled DNA was isolated by polyacrylamide gel electrophoresis as described in section
2.6.2. The amount of protein used per reaction ranged from 1 to 2 jlg and 8 fmol of the labelled DNA was added if not specified otherwise. 2 jlg of poly (di-dC) was added to prevent non-specific binding of proteins to the DNA. Reactions in footprint buffer
(section 2.2.2) were carried out in a total volume of 12 j.tl at room temperature for 20 min. 10 j.tl of the reaction mixture was loaded on a pre-electrophoresed 5% (w/v) polyacrylamide gel (98.8: 1.2 acrylamide: bis-acrylamide; section 2.2.2) containing 5%
(v/v) glycerol and TBE buffer and the DNA-protein complexes were separated from free
DNA by electrophoresis in TBE buffer containing 2-mercaptoethanol (8.6 mM) at 10
Vcm-1 for 2 hat 4"C. DNA migration was monitored by the addition of bromophenol blue and xylene cyanol to the free lane.
Gels were dried under vacuum and radioactivity was scanned by Phosphoimager analysis to quantify the intensity of the retarded species as a DNA-protein complex in comparison with the free DNA. Alternatively, gels were autoradiographed overnight at
-70°C with two DuPont Reflection™ intensifying screeens and Reflection™
Autoradiography Film NEF-. Densitometric scanning was performed for the THF effect analysis by Imaging Densitometer (Bio-Rad).
In competition experiments, the unlabelled competing DNA was added to the protein extract and preincubated for 10 min before the addition of end-labelled DNA.
2.8.3 Footprinting analysis
Cu2+fphenanthroline footprinting In situ footprinting assays were performed as described in Sigman et al. ( 1991) with modifications. After binding activities were determined by EMSA (section 2.8.2), forty fmol of 32 P end-labelled DNA
72 Materials & Methods was incubated with enough protein to completely complex the DNA in footprint buffer
(section 2.2.2) to a total volume of 36 J.ll and incubation for 15 min at room temperature.
After the EMSA was performed, the whole gel was immerged into 200 ml of Tris-HCl, pH 8.0 and was added with 20 ml of freshly prepared solution containing 2 mM phenanthroline and 0.45 mM CuS04 and 20 rnl of 58 mM 3-mercaptopropionic acid. The reaction was continued for 10 min at room temperature and then quenched with 20 rnl of
28 mM 2,9-dimethylphenanthroline for 2 min. The gel was rinsed briefly with distilled water and exposed to X-ray film for 10 min at room temperature. The shifted bands were cut from gel and the gel-slices were eluted overnight at 42'C in solution containing 0.5 M ammonium acetate and 1 mM EDTA. The eluted DNA was ethanol precipitated, resuspended in 10 J.ll of sequencing gel-loading dye (section 2.2.2) and was electrophoresed with G+A sequencing ladder on an 8% sequencing gel (section 2.6.2) after being heated to 95'C for 5 min. A G+A sequencing ladder was prepared from 40 fmol of the same fragment as described in Sam brook eta!. ( 1989).
DNasel Footprinting Analysis The footprint assay mixture (50 J.ll final volume) contained 2-3 ng (-20,000- 30,000 cpm) of 32p end-labelled DNA fragment, 5
J.lg of poly dl-dC, and 15 to 30 J.lg of protein extract in the footprint buffer as described in section 2.8.2. Bovine serum albumin (10 J.tg/ J.ll) was added to give the same amount of protein in each reaction. The DNA fragment used was end-labelled with [a-32P]dATP and isolated by polyacrylamide gel electrophoresis (section 2.6.2). After 20 min incubation at room temperature, freshly prepared DNasel (4.5 units) diluted with
Ca2+fMg2+ solution ( 10 mM) was added and the DNA digestion was proceeded for 1 min at room temperature. The reaction was stopped by adding 140 J.ll of stop solution containing 192 mM sodium acetate, 32 mM EDT A, 0.14% SDS and 64 J.lg!ml yeast
RNA. DNA fragments were extracted once with phenol/chloroform (1: I), and precipitated with ethanol and 0.3 M sodium acetate. The pellet was rinsed with 95% (v/v) ethanol and resuspended in 4 J.ll of sequencing gel-loading dye (section 2.2.2). The DNA fragments were denatured for 1 min at 95'C prior to electrophoresis on an 8% sequencing
73 Materials & Methods gel. A G+A sequencing ladder was prepared from the same fragment used in footprinting reaction as described in Sambrook eta!. ( 1989).
2.9 B-Galactosidase assay
The method of Rose and Botstein (1983) was used for the assay of B galactosidase in yeast. Culture (50 ml) was grown to an OD60o of 0.4-0.5, harvested by centrifugation for 5 min at 5000 x g in SS34 centrifuge tube, resuspended in 0.5 ml of cold breakage buffer (100 mM Tris-HCl pH 8.0, 1 mM DTT, 20% (v/v) glycerol), and transferred to an Eppendorf tube. 12.5 ftl of PMSF (40 mM) and 0.3 g of acid-washed glass beads were added to the tube and homogenized for 1 min. For the assay, 900 ftl Z buffer (section 2.2.3) and 20-100 ftl of cell extract was pre-incubated at 29'C before the addition of 200 fll ONPG (4 mg/ml) to the assay tube or 200 ftl HzO to the control tubes.
Yellow colour developed in the presence of B-galactosidase. The reaction was terminated with 500 ftl of 1M NazC03 and the absorbance was measured at 420 nm.
Protein concentration of the extracts was measured using the dye-binding assay of
Bradford (1976). A protein standard curve was constructed with different amount of bovine serum albumin by adding 200 fll Bradford's reagent, making up to 1 ml with sterile water and measuring the absorbance at 595 nm. The protein content in 20 ftl of the extract was then determined.
All assays and protein readings were performed in duplicate. Specific activities of
B-galactosidase (nmol ONPG hydrolysed/min/mg protein) were expressed according to the formula:
Specific Activity = 1.7 x OD420 I (0.0045 x protein concentration x extract volume x time in minutes).
Where OD420 is the optical density of o-nitrophenol, at 420 nm. The factor 1.7 is for the correction of the reaction volume and 0.0045 is the optical density of a 1 ftM solution of o-nitrophenol. Protein concentration is expressed as mg/ml and extract volume is the volume (ml) assayed.
74 Materials & Methods
2.10 Computer analysis
Graphics were drawn on "MacDraw®Pro l.Ovl". The Microsoft program
"Word® version 5.la" was used for word processing. Graphs were produced using
"CA-Cricket Graphiii v 1.0". Plasmids were drawn using the Macintosh program
"MacPlasmap version l.S2iD". Phosphoimaging to quantify radiolabelled probes and to produce densitometry was analyzed with "Multi-Analyst (version 1.0.1; Bio-Rad
Laboratories, Inc.)". The autoradiographic images were edited by "Adobe® Photoshop® v5.0" for Macintosh.
Basic DNA and protein sequence analysis (including DNA restriction mapping, open reading frame mapping) and uploading/downloading of sequences to the AN GIS was done using "DNA strider™ vl.O". The comprehensive DNA and protein computer analyses were made available through the internet from: the Australian National Genomic
Information Service (WebANGIS; University of Sydney, Australia; http://www.angis.su.oz.au); Saccharomyces Genome Database (SGD; Stanford
University, U.S.A.; http://genome-www.stanford.edu/Saccharomyces); Yeast Protein
Database (YPDTM; Proteome Inc.), a database that contains information on the physical and functional properties of all the proteins of the yeast Saccharomyces cerevisiae
(http://quest7. proteome.com/YPDhome.htmi).
75 Regulation of GCV genes
Chapter 3: ANALYSIS OF THE REGULATION OF G C V GENES IN VIVO
3.1 Regulation of GCV genes by different nutrients.
3.1.1 Introduction.
The glycine decarboxylase multienzyme complex (GDC; EC 2.1.2.10) is a mitochondrial enzyme in eukaryotes which is encoded by three GDC-specific genes
(CCV 1, T -subunit; GCV2, P-subunit; and GCV3, H-subunit) and the LPDJ gene (L- subunit; lipoamide dehydrogenase) which is also a subunit of at least three other multienzyme complexes (Dickinson and Dawes, 1992; Dickinson et al., 1986). The
GDC catalyzes the following reversible reaction (Koichi and Kikuchi, 1974):
Glycine+ THF + NAD+ <--> 5,lO-CH2-THF + C02 + NH4+ + NADH
The GDC serves as the entry point for the use of glycine as an one-carbon donor. Previous studies have shown that, in the absence of serine, glycine can supply all cellular one-carbon requirements inS. cerevisiae (McKenzie and Jones, 1977; Ogur et al., 1977;
Zelikson and Luzzati, 1977) and when lacking GDC activity, the cell's ability to utilise glycine as a sole one-carbon source is abolished in a serl background (Sinclair and
Dawes, 1995). Glycine enters one-carbon metabolism via GDC by converting uncharged
THF to 5,10-CH2-THF containing the one-carbon functional group (Ogur eta!., 1977;
Pasternack et al., 1992). In addition, the GDC is a part of the serine-glycine interconversion pathway together with serine hydroxymethyltransferase (SHMT) (Figure
3.1), and thus participates in the metabolism of various essential two- and three-carbon compounds (Pasternack et al., 1994a). The reaction catalysed by the GDC also enables yeast cells to use glycine as sole nitrogen source (Sinclair and Dawes, 1995). Glycine can be classified as a poor N-source since the only end-product of glycine catabolism by the GDC is ammonia (section 1.4.1).
76 Regulation of GCV genes
In this chapter, it will be shown that there is a novel system for the coordinate regulation of GCV genes under different nutritional conditions and the cis-acting elements in the genes' promoters mediating these controls have been identified. The nature of the transcriptional regulation systems governing this control are discussed as well as the possible relationship of other well-known transcription systems with the regulation of
GCV gene expression.
Glycine Serine NAD+ THF
SHMT
NADH+H+ 5,10-CHrTHF Glycine
Figure 3.1 The serine-glycine interconversion pathway. Serine and glycine catabolism is linked by THF and S,lO-CH2-THF. Glycine cleavage by the GDC produces S,IO-CH2-THF which is then used for the production of serine together with another glycine molecule by SHMT. Likewise, serine catabolism by SHMT produces glycine which can be subsequently degraded by the GDC's catalytic activity.
3.1.2 Structure of promoter region of GCV genes.
The initiation stage of mRNA synthesis is a major site for the regulation of gene expression (section 1.5) and the mechanism of mRNA synthesis is highly ordered in all organisms. In S. cerevisiae, initiation is governed by DNA sequence elements comprising several functional classes. Core elements define the site for assembly of the transcription preinitiation complex (PIC) and include a TAT A sequence and an initiator sequence (Inr). Regulatory elements are gene-specific sequences which regulate the rate of transcription and are generally located upstream of the core elements. These include upstream activation sequences (UAS) and upstream repression sequences (URS), which serve as binding sites for activators and repressors of transcription, respectively. Genes subject to a common control mechanism share common upstream elements whereas non- coordinately regulated genes contain different upstream elements (Struhl, 1989). In
77 Regulation ofGCV genes
Yeast UAS's are analogous to mammalian enhancer elements in that they can function in both orientations at longer and more variable distances from the mRNA start site compared to core elements. However, unlike enhancer elements, UAS's do not
activate transcription when located downstream of the mRNA start site. This property of
U AS does not appear to be intrinsic because an U AS and its binding factors from yeast function downstream of mammalian promoters in mammalian cells (Webster et al.,
1988). Therefore, whether it is an enhancer or a UAS depends on the context in which it is functioning and there is an evidence that yeast genes can be regulated by intragenic
DNA sequence elements (Sinclair et al., 1994).
It might be expected that genes encoding the GDC are co-regulated and it has been suggested that genes subject to a common control mechanism would share common upstream elements (Struhl, 1989). Figure 3.2 shows the identification of some potential cis-elements in the upstream and downstream regions (-500 to +100 bp relative to the start codon) of the GCV genes. Searches for the potential regulatory factor binding sites were performed with PatternSearch v .1.1 (Heinemeyer et al., 1998) and Matlnspector
v.2.2 (Quandt et al., 1995) programs, which are available through the World Wide Web 1•
For Matlnspector 2.2, the parameters for core similarity and matrix similarity were 0.75 and 0.85, respectively. The function of each potential element found through this
analysis is discussed briefly below.
It is notable that there are at least three potential GCN4 sites (consensus: 5'-
TGACTC-3') in the upstream region of all three genes. Gcn4p (which may bind to these putative GCN4 sites) is the positive regulator of the general control of amino acid biosynthesis (section 1.5.3). Since the GDC can participate in glycine biosynthesis due to the reversibility of its reaction, GCN4 is a good candidate as a regulator of these genes.
Moreover, in the course of this thesis research it was implied that there is some
involvement of Gcn4p in GCV3 gene expression (Nagaraj an and Storms, 1997).
1 Pattern Search v.l.l; http://bioinfo. weizmann.ac.il/cgi-bin/palSearch/patsearch.pl Matlnspector v.2.2; http://transfac.gbf.de/cgi-bin/matSearch/matsearch.pl
78 Regulation ofGCV genes
GCV1 -500 GAAGATACCA AAAGCCAAAT AGAAGGCAAC AATACGAACA ACAAAATACG C~AfGT~TA TGGGATF-TCG PHO ABF1 -430 AAGCTGAAAC AATGGACTTG GAG'AAAGATA GATATTACGA TCTGTCATGA GGTCCTTGCC TTCTATATCT
-360 GCGCCACTTT TAATCGGCTC GATGCGACTA ACATCCGTAT T~CCCACAC J'}AGCT~ATTG ATTTTTTTTT RAP1 -290 TGCATTCTTG GATCTCGATG GGCGCTTTTA CTCCATTAGA ACCATGCTCA C~TG AGTTCTTACT GCN4 GCR -220 TAGAGCTGGT CATAGATATC ACCTATTTTG TTCATCGCCG TGACTTCTTT CGGCAGGGCG ACTCTGCTCA GCN4 GCN4 -150 GGAGCCCTGA CTCGTGTTGC CTCGAGTAAA CGGTATGTCA AAAAGAATAG GTCCAAATAT CATATATATA GCN4 -80 GCACCTTG~ CCCAAAC~ TECAATT~A GTACCCGTAG CTCACATATG TAGAAAGAGC TTTGCCTGGC RAP1 HAP21 14 -10 ACAAGCAATA ATGTCfATAA TCAAAAAAAT TGTfTTTAAG AGATTCAACT CAACf~TGAA AAAAACTGCT I +1 BAS2 GCN4 BAS2 +60 CTTCATGACC TTCATGTGTC ATTAGGCGGT ACAATGGTAC
GCV2 -500 GCCATTACAG TTTAACTGAT CTTGATTTGA CT~TTGAAAT CTCTAAI::CCG AATTCAACTC CCTGAGTTTA BAS2 -430 CTTCTGTTTG GCCTTATTTG TGGATTTCCT CTAACAGTTT GATGCTCGCA TTGGTTTGGC ACAATCCCAA.
-360 ATTGCTTCAG AA.TTCTAA.TC TTATGAA.ATA ACAA.TCATGT TACCCGGTTG AATCGfTACC CG! -80 CTGTATTGAT TGCTGCGCGA TCTAA.CCATC AACTAACCAC AG~TATTGGIAAAAACGTTT CCTAATACCA HAP2;314 -10 ~TAAC ATGCTTAGGA CAAGAGTGAC TGCTCTCCTT TGTAGGGCTA CTGTC.;_GGTC AAGCA(¢CAAT GCR +1 GCN4 HAP2 /3 I 4 +60 ~GTTTCAT TAGCGAGGAC TAGATCATTC CATTCTCAAT GCV3 -500 GTCCTTAAGA GTTCTCGAGT CTCTGGGTCC ATTCCTTGAA. AACAA.ACACA GACGATTCTT CATCAGACTT -430 ATGAGCGAA.C TGCCCAGTCT TCAJV\GCGAT CATCTTGAGG CACTGAA.GCC TATCTGCCTG GATCCGGCAA -360 GAA.GTTCCCT TGGTTTCCAA. ACGCTAJV\GT TTCTCATTAT GTTTAGACCC CCAGTGCAGG ACACTGTTCG -290 CGACCTGCTG CATCAGCTAA AGCAAGAAGA TGAAGGCTTA CACAA.GCAGT GCGATTCACT GCTTGACAGG -220 CTAAA~TGAT CACAACCATT ACATACATCA TATATACGCA TGTGTAGGGT ~4TCAA CTTGCAAATC GCR -150 AATGTTTTAT CTATCATTCT GTGCTATAGG GACCTCGCAA TTTTGACACT TCCGTAAGGA GTCATTCAGC GATA GCN4 -80 GGCGGAGTCA CTTTCGCTAA CTCTTTCTTC TATATATACA GAfCAACAAJ' CAAGTATCTA ATGEGAAAGG GCN4 HAPU3 I 4 BAS2 -10 GAATCTCCACIATGAAGCATA CCACATCGAC AATGTTACGC ACTACTAGAC TATGGACCAC CCGCATGCCC +1 +70 GCTGTGAGCA AATTGTTTTT GAGAAACAGC TCCGGCAA.TG 79 Regulation ofGCV genes Curiously, the transcriptional activator Bas1p also binds to the same TGACTC site. This is involved in the transcriptional control of genes for purine synthesis (section 1.5.3). Since the GDC may play a role in purine synthesis via one-carbon metabolism (Pasternack et al., 1994a), Bas I p is another candidate for a regulator of the expression of GCV genes. In fact, there is some indication that there may be some role for Bas 1p in the regulation of GCVJ (Denis and Daignan-Fornier, 1998). Several Bas2p-binding sites were also found in the promoters of the GCV genes. Bas2p (also called Pho2p) is known to be required for optimal gene expression mediated by Bas I p (section 1.5.3). However, a study of ADE genes revealed that the DNA binding activity of Bas2p was not required for activation by Bas 1p (Zhang et al., 1997) and moreover, the putative Bas2p-binding sites of GCV genes in Figure 3.2 were found by homology between the BAS2 site found in the upstream of the PH05 and HO genes which are involved in phosphate homeostasis and mating type in the cell; it appears that these elements are unlikely to have significant function in GCV genes. An element (consensus; 5'-GATAAG-3') responsible for GAT A-factor dependent nitrogen catabolite repression (NCR; section 1.4.3) was found in the GCV2 and GCV3 promoter regions. From saturation mutagenesis studies and gel-mobility shift assays, it was shown that GAT A factors bind to an element with the GAT AA sequence at its core (Bysani eta/., 1991; Cunningham eta/., 1996). There are no such sequences found in GCVJ, however, there are two 5'-GATA-3' sequences oriented head-to-tail at -404 bp and two other GAT A sequences are found ( -497 and -365) in the promoter region of GCVJ. Naturally occurring binding sites in promoters are often dissimilar to the optimal binding sequence for the cognate transcription factor (Kunzler eta/., 1996; Luche et al., 1990; Shore and Nasmyth, 1987) and this sequence flexibility of eukaryotic cis-elements makes it difficult to distinguish whether a given homologous sequence element is functional. Binding motifs for the HAP complex were also found in the upstream regions of the GCV genes. The HAP complex (also called CCAAT box-binding factor) is a heteromeric complex composed of at least four subunits, Hap2p, Hap3p, Hap4p and 80 Regulation ofGCV genes Hap5p, and mutations in any of the HAP2/3!4 genes resulted in blocking of the expression of genes encoding mitochondrial proteins required for the growth on lactate medium (Forsburg and Guarente, 1989; Hahn et al., 1988; McNabb et al., 1995; Pinkham and Guarente, 1985). Hap2p, Hap3p and Hap5p are required for DNA-binding activity, while Hap4p provides a transcriptional activation domain (McNabb et al., 1995; Olesen and Guarente, 1990). The major role of this complex is carbon-dependent regulation of several genes implicated in respiration (de Winde and Grivell, 1993), however, it was also shown that the HAP complex affects the expression of some genes involved in nitrogen metabolism (Bowman et al., 1992; Dang et al., 1996; Dang et al., 1996), and that this complex stimulates expression of several genes involved in mitochondrial biogenesis at the transcription level (Dang eta!., 1994 ). The binding sites for Raplp, Gcr1p, Abfl and Reblp were also found in the upstream regions of GCV genes. The repressor-activator protein Rap I p activates transcription of glycolytic and translational component genes interdependently with Gcrl p by forming heteromeric complexes (Santangelo and Tornow, 1990; Tornow et al., 1993). Gcr1p recognizes a sequence called the CT box, which has shown to primarily function in glycolytic promoters (Bitter et al., 1991). Depending on the sequence context of its binding site, Rap I p is capable of controlling many diverse cellular functions such as control of telomere length and activation or repression of transcription by interacting with other cellular regulatory factors (Baker, 1991; Shore, 1994 ). Binding sites for the other abundant multifunctional proteins, Abfl p and Reb 1p are also found in many genes, particularly those in pathways of translation, glycolysis and cell differentiation (Della Seta et al., 1990; Graham and Chambers, 1994; Packham eta!., 1996). While this search for known regulatory motifs is interesting, there are many examples known of control sequences being present in the promoters of eukaryotic genes, but lacking function. Therefore, there is a need for a more detailed analysis of factors affecting expression of the GCV genes and to identify the functional elements in the gene. 81 Regulation oJGCV genes 3.1.3. Regulation of the GC V genes in S. cerevisiae The molecular studies on the regulation of the gcv operon (section 1.3.2) in E. coli have provided some insights into the regulation of the eukaryotic glycine cleavage system, the molecular mechanisms of transcription initiation and its regulation in E. coli are qualitatively different from that of eukaryotes. The properties of promoters in S. cerevisiae are very similar to those found in higher eukaryotes and principles of transcriptional regulation in yeast have proven broadly relevant to higher organisms. A. B. 1000 ~ = • Glycine E = 1 ·e 800 oo Serine -E"" - D ;:, E 0 ::,: E 900 s sE 600 .:::- .:::- ·;: ·;: t) 600 -«: u 400 u < ·w<.;: .:2 ~ 300 ·c 200 c. 0c. "' "' 0 -7 -6 -5 -4 -3 -2 -1 0 Log1oconcentration (M) Figure 3.3. Expression of GCV2 under different nutritional conditions. (A) Cells transformed with pRH I were grown to OD600 of 0.4- 0.5 in different media (section 2.1.7) then harvested for ~-galactosidase assay as described in section 2.9. (B) The effect of glycine and serine on GCV2 expression. Cells were grown in Dmin to an OD600 of0.9- 1.0, then the culture was transferred to fresh Dmin containing different concentration of glycine or serine. After a further incubation for 2 h, the cells were collected for ~-galactosidase assay. The final OD600 of the culture was 0.4-0.5. Errors were less than 20%. To study the regulation of genes encoding the GDC, plasmid pRHl was constructed from plasmid pGSD2.5 (Sinclair et a!., 1996) containing the intact GCV2 gene. The EcoRI-Xhai fragment of pGSD2.5 spanning -351 to 378 bp of GCV2 was isolated and fused in-frame upstream of the lacZ gene in the integrative S. cerevisiae/E. coli shuttle vector, Ylp358 (Myers eta!., 1986). pRHI and all the lacZ fusion constructs 82 Regulation ofGCV genes used in this study were sequenced to confirm the accuracy of their construction. The pRHl (and also other lacZ fusions constructed in this study) was linearised by cutting at a unique Stui site in URA3 to target integration to the ura3 locus of the haploid yeast strain BWG 1-7 A (Y3). Transformants resulting from recombination were selected on media lacking uracil (section 2.5.2) and Southern analysis (section 2.7.4) was carried out using a lacZ probe to choose singly integrated constructs. The transformants were then grown under different nutritional conditions for B-galactosidase assays as described in section 2.9 to monitor the expression pattern of the GCV2 gene (Figure 3.3). Glycine and serine were of particular interest because of the GDC's role in the reversible interconversion of glycine and serine (Figure 3.1). Figure 3.3A shows that the GCV2 gene can be regulated over an 100-fold range, of which about 20-fold is due to repression in rich medium, and 5- to 7-fold due to glycine induction. The GCV2 gene showed a very low level of expression in complete media (YEPD and YEPL). Interestingly, the level of expression was increased approximately two-fold in cells grown in YEPL relative to the level observed in cells grown in YEPD, consistent with previous results (Sinclair eta/., 1996): this may indicate a role for the HAP complexes. The LPDJ gene encoding the L-subunit of GDC is regulated by the HAP complex (Bowman eta!., 1992), and also the function of the HAP complex is not restricted solely to genes involved in carbon metabolism and respiratory function, since it is involved in the regulation of others including nitrogen metabolism-related genes (Dang eta!., 1996; Dang eta!., 1996). Its potential cognate binding sites are found in the GCV genes, although they are all located 3' of the putative TATA box, and some are within the coding sequences (Figure 3.2). It is known that some yeast genes are regulated by intragenic cis-elements (Sinclair eta/., 1994) and further study is required to see whether the GCV genes are regulated by the HAP complex. In Dmin medium, the level of expression was increased about 10- to 20-fold relative to that in complete media. When glycine was added to Dmin (Dmin+gly) at a final concentration of 10 mM, the expression level was further increased by 3- to 4-fold. However, when L-serine was added instead of glycine to Dmin, there was no significant 83 Regulation ofGCV genes increase in expression. The highest level of expression (seven-fold increase from Dmin) was observed in cells grown in GL Ymin where glycine is the sole nitrogen source. Further investigation of this glycine response was performed by monitoring GCV2 expression levels in cells grown in Dmin containing different concentrations of glycine or serine (Figure 3.3 B). Cells were grown for two hours to induce expression of the gene; this time was determined to be optimal for gene induction (D. Sinclair. UNSW, pers. comm.), and chosen to minimise glycine/serine concentration changes caused by cell utilisation. It was shown that GCV2 transcriptional activity was low at external glycine concentrations below 50 ~-tM and an increase in expression was observed over a relatively narrow range of external glycine concentration (between 50 ~-tM and I 0 rnM). Furthermore, the intracellular glycine concentration and the expression of the GCV2 gene varied in parallel under these conditions (Sinclair et al., 1996). The intracellular concentration of glycine did not increase until the initial medium concentration was 50 ~-tM presumably reflecting the level of cellular synthesis of glycine. Above this level, increases in extracellular glycine led to an increase in both intracellular glycine and the rate of expression of the GCV2 gene. This threshold effect is presumably a response to excess glycine in the medium. However, there was no induction of the gene by serine despite its uptake into the cell (intracellular glycine and serine concentrations were determined in collaboration with D. Sinclair, UNSW). An additional experiment was performed using available glycine structural analogues and glycine receptor (GlyR) agonists (Davidoff et al., 1967), to give an insight into the role of glycine on the expression of GCV2 (Figure 3.4). Agonists are molecules which trigger an effect in target cells after binding to their receptors. Expression of the S. cerevisiae arginase gene (CARl) was shown to be induced by arginine or the arginine analogue homoarginine (Sumrada eta/., 1982; Whitney and Magasanik, 1973). However, all the glycine analogues tested did not show any significant effect on the expression of the GCV2 gene. Therefore, there is no evidence for glycine being a direct effector/signal for the expression of GCV2. 84 Regulation ofGCV genes CH2 CH2 CH2 NH /""- / ""- /o, / NH + coo- NH3+ c CH3 NH3+ ""/c ' OH 3 II 0 ~ Glycine Glycine methylester Glycine hydroxamate /cH\ /coo- NH3+ CH2 Taurine ~-Alanine Glycine Glycine Drnin Glycine methylester hydroxamatc Taurine B-a\anine Specific Activity (nmol/mg/min) 204 ± 47 743 ± 194 193 ± 38 161 ± 37 232 ±59 200 ± 25 Figure 3.4 Effect of glycine analogues on the expression of the GCV2 gene. The diagram above shows the structures of glycine and its analogues. Cells transformed with pRHl were grown in Dmin or Dmin containing a different compound at a final concentration of 10 mM and then harvested for ~-galactosidase assay as described in section 2.9. To investigate whether this glycine response is coordinately regulated among genes encoding the other subunits of the GDC, GCVJ-lacZ and GCV3-lacZ fusions were also constructed. The -1026 to +96 (Sphi-Kpni) fragment of GCVI was isolated from YEpl3::GCVI (section 2.1.5) and cloned into Yip357R (Myers eta/., 1986) in frame with lacZ. The GCV3 gene sequence from -410 to +1328 was amplified from genomic DNA using PCR primers 5'-tcaaagcgatcatcttgaggc-3' (-410 to -390) and 5' acttcttgcttgttatgttgatagc-3' (+ 1303 to + 1328). The BarnHI fragment of GCV3 ( -371 to +1291) was then cloned into pTZ19 (section 2.1.5). Subsequently, the GCV3 BarnHI to Sphi (-371 to +53) fragment was subcloned into Ylp356 (Myers et al., 1986) and fused to the lacZ reporter gene. The LPDI-lacZ fusion reporter (pDS 1) containing -764 to +697 of LPDI gene has been constructed by and obtained from D. Sinclair, UNSW (Sinclair et al., 1994). The extent of glycine control over GCVJ, GCV2, GCV3 and LPDI expression in the same strain using these constructs is shown in Figure 3.5. The GDC-specific genes GCV1 and GCV3 showed similar glycine responses to that of GCV2. This is in agreement 85 Regulation ofGCV genes with the results observed by other investigators using multicopy GCV-lacZ constructs (McNeil et al., 1997; Nagarajan and Storms, 1997). LPDJ showed a fairly high level of expression in cells grown in minimal medium with the addition of glycine having no significant effect on expression. This is in agreement with the result of Sinclair et al. (1996), who showed that the LPDJ transcript levels were not changed significantly in cells grown in the presence or absence of glycine . These results are not surprising since in yeast LP D I encodes lipoamide dehydrogenase which is a subunit for at least three other multienzyme complexes including pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and the branched chain 2-oxoacid dehydrogenase (Dickinson and Dawes, 1992; Dickinson et al., 1986). 1200~------~ D Dmin D Dmin + I 0 mM glycine GCV2 GCVI GCV3 LPDJ Figure 3.5. The response to glycine of expression of the four genes encoding the GDC components. Upstream sequences of the genes encoding the P-subunit (GCV2), the T-subunit (GCV 1), the H-subunit (GCV3) and the L-subunit (LPDI) were fused in-frame with the /acZ reporter gene and each transformed separately in to strain Y3 forB-galactosidase assays. Cells were initially grown to an 0D6QO of 1.0 in Dmin medium and shifted to fresh Dmin with and without I 0 mM glycine for 2 h. B-galactosidase assays were performed in triplicate as described in section 2.9. The coordinate nature of regulation of GCV genes was also extended to other forms of regulation. Expression levels of GCV genes were examined in glucose minimal media containing ammonia (Dmin), glycine (GL Ymin), L-proline (PROmin), or L- 86 Regulation ofGCV genes glutamine (GLNmin) as sole nitrogen source as well as in glucose complete medium, YEPD (Figure 3.6). A. B. c. 500 ~ GCVJ GCV3 <= <= E E 400 ~I E -;§'"' -0 c sE sE 300 .':' c :~ ;: u 200 ~ ~ u u ;;::" <= u ·::; "c. "c. 100 VJ "' ·!;- 0 o/ ¢"' ~'> . "' ;\) '\!' },>..:.; 1'1.0 ~o/' 4.4} 0 ~ ()Y Figure 3.6. Coordinated regulation of CCV genes. Cells were grown to OD6Qo of 0.4 - 0.5 under different conditions and harvested for ~-galactosidase assay as described in section 2.9. Expression levels of GCV2 (A), GCVJ (B) and GCV3 (C) are shown. 87 Regulation ofGCV genes Very similar patterns of expression were observed between the three CCV genes under all conditions tested. As expected, the highest level of all genes' expression was observed in cells grown in GL Ymin. It also appears that there is some form of nitrogen catabolite repression (NCR; section 1.4.2), since cells grown in poor nitrogen sources (L-proline and glycine) showed higher levels of CCV expression than in rich nitrogen source (L-glutamine). The GDC catalyses the breakdown of glycine to produce NH4+, thus it can be involved in nitrogen metabolism and the NCR is a characteristic of most nitrogen catabolic genes. When cells were grown in YEPD, the expression of all three CCV genes was at its lowest. There are at least two possible explanation for this regulation. First, the GDC complex plays an important role in one-carbon metabolism by generating 5, l0-CH2-THF which serves as an intermediate of one-carbon metabolism by mobilisation and utilisation of the functional one-carbon units (Song and Rabinowitz, 1993). Complete medium is rich in the products of one-carbon metabolism such as purines, choline, thymidylate and pantothenate as well as serine, whereas minimal media lacked these compounds. Cells grown in minimal media presumably need to induce the genes to cope with this demand. However, addition of the end-products of one-carbon metabolism to minimal medium (Dmin) separately or in combination did not reduce the expression of CCV2 (M. Piper, UNSW, pers. comm.). Secondly, it may simply be due to NCR since YEPD medium contains a mixture of good nitrogen sources. Expression in cells grown in YEPD was reduced four- to five fold compared to GLNmin grown cells, which may reflect the additive effect of the presence of multiple good nitrogen sources in YEPD. To test this hypothesis, good nitrogen sources were added to Dmin separately or in combinations. Figure 3.7 shows that CCV2 expression in a medium with L-asparagine as sole nitrogen source was as low as GLNmin. L-asparagine is considered as a good nitrogen source in S. cerevisiae (Cooper, 1982). Furthermore, when the good nitrogen sources were added in combination, the level was even lower, approaching that of YEPD grown cells. Therefore, the effect of complex medium on the expression of CCV genes is, at least 88 Regulation ofGCV genes partially, due to the presence of good nitrogen sources. A similar situation can be found in the regulation of the GAP 1 gene encoding general amino acid permease (Soussi- Boudekou eta/., 1997). It was shown that the expression of this gene was 10- to 70-fold lower in a medium with a mixture of good nitrogen sources than in media containing a single good nitrogen source, determined by assay for expression of GAP 1-lacZ carried on a low-copy-number plasmid. E" bD ~ 200 ~ ·;;:c .B ~ u 100 ·uc.= u c. "' 0 Cl .E" " "E " .E" c. I oJ 0 i z ...l (IJ ~" + >< 0 ~ ~ i' + z -= + 2 ~ ~" + 2-= Figure 3.7. Effect of different nitrogen sources on the expression of GCV2. Cells were grown in media containing different nitrogen sources and 13-galactosidase assays were performed as in section 2.9. Ammonia, L-glutamine, or L-asparagine were the sole nitrogen sources for Dmin, GLNmin, and ASNmin, respectively (section 2.1.7). The (Gin + Asn)min and (Gin + Asn + :-.IH4+)min media contained equal proportions of GLNmin and ASNmin, or Dmin, GLNmin and ASNmin, respectively. 3.2 Promoter analysis of GCV genes. 3.2.1. Identification of two potential regulatory elements To localise the control regions for the glycine response and YEPD-repression (or nitrogen regulation), deletion analysis of the upstream region of the GCV2 gene was 89 Regulation ofGCV genes performed. Plasmid pGSD2.5 used to prepare plasmid pRH1 was also used for the construction of pRH2 ( 1.37 kb Kpni/Xbai GCV2 fragment). Plasmid pRH3 contains a 585 bp EcoRVXbai GCV2 fragment (-206 to 378 bp) isolated from pGSD2.5~5 (Sinclair eta!., 1996). Each GCV2 fragment was fused in-frame upstream of the lacZ gene in Ylp358 (Myers eta!., 1986). A PCR-based technique with pRH1 as a template was employed to make further deletion constructs, pRH4, pRHS and pRH6. For each construct, common oligonucleotides (GCV2-1R; section 2.1.4) and a set of oligonucleotides harbouring EcoRI recognition sequences was introduced to use as a restriction site (GCV2-2F for pRH4, 4F for pRHS, and SF for pRH6; section 2.1.4). Yip358 as well as the PCR products were then cut with EcoRVXbai and ligated to yield the constructs. Site-directed mutagenesis of two potential GCN4 sites (-312 and -291; Figure 3.2) of GCV2 were performed (section 2.3.6) using pRHl as a template, and oligonucleotides designed to mutate the potential GCN4 sites to Kpnl restriction sites (MGCN4A and MGCN4B; section 2.1.4). The PCR-products were cloned into Ylp358 after cutting with EcoRVXbai. The constructs with the GCN4 site mutated at -312 and -291 were designated pRH7 and pRH8 respectively and a construct with both GCN4 sites mutated (pRH9) was also made in the same way using the plasmid pRH7 as a template and an oligonucleotide, MGCN4B. Further 5'-extending deletions were constructed from pRH7 and pRH8 by cloning the 690 bp and 669 bp Kpnl!Xbal fragments into Ylp358, to produce pRHlO and pRHll respectively. The unidirectional deletion constructs were used to localise the region that is responsible for the glycine response of the GCV2. Each construct was transformed into yeast strain Y3 and single copy integrants were chosen with Southern analysis for B galactosidase assay. It was observed that the glycine response was completely abolished on deletion of sequences to -267 (pRH4; Figure 3.8 A) and the effect of glycine induction discussed in section 3.1.3 is actually due to a loss of repression of GCV2 since deletion to -267 led to a constitutively higher level of expression (Figure 3.8). 90 Regulation ofGCV genes A. -gly +gly (fold) -I kb c::« I I I I I I I I I I I I I M • RH2 247 ±9 1190 ± 152 (4.82) -351 I I I I I I I I I I I I I I RHI 204 ±47 734 ± 194 (3.60) "*' • -313 I I I I I I I I I I I I M • RHIO 213±62 625 ± 118 (2.93) -289 I I I I I I I I I I 989 ± 246 (2.20) "*' • RHll 450 ± 76 -267,CJIJIJI~ILIJIJI~~~~=-~~~~N • RH4 1254± 350 1231 ± 189(0.98) D GCM L-51 • RH5 1406 ± 459 1420± 331 (1.01) ~ GATA -205 fi.i 1562 ± 232 (0.97) • TATABox • RH3 1609±185 -1941 • RH6 1505 =260 1339 ± 147 (0.89) 1800 B. RHI D io 0 RH2 = • .§ RH3 0 OJl E 1200 RH4 e :::0 0 ! ~ .·;;., u 600 ~ ~ ~ u " r/l"' 0 -7 -6 -5 -4 ·3 -t -1 0 Logwglycine concentration (M) Figure 3.8. Deletion analysis of the GCV2 promoter to localise sequences involved in the glycine response. A. Various deletion constructs were transformed into strain Y3 as single integrated copies and cells were grown and assayed for B-galactosidase activity. The extent of the deletions is presented on the left of the diagram with the location of potential GCN4 sites, the GATA element and the TATA box as indicated. Fold-induction of expression from Dmin (-gly) to Dmin supplemented with 10 mM glycine (+gly) is indicated in brackets. B. The glycine-response of GCV2 gene is mediated by repression. The glycine response of four representative deletion constructs outlined above was determined at differing concentrations of glycine in the external medium. B-galactosidase assays were performed as described in the legend to Figure 3.3B. Open squares represent the full length construct in pRH I, closed squares pRH2, open circles pRH3 and closed circles pRH4. Errors were less than 20'7<. 91 Regulation ofGCV genes It was also notable that repression of the GCV2 gene in the absence of glycine in the medium was partially relieved about two-fold on deletion of sequences to -289 (pRHll; Figure 3.8 A). It appeared therefore, that there are additional important sequences between -313 and -289 affecting GCV2 repression. There are 3 potential GCN4 sites between -313 and -267, which may bind Gcn4p, the transcriptional activator mediating general control of amino acid biosynthesis (section 1.5.3). However, its nature as a repressor has not been demonstrated (Hinnebusch, 1992). To locate the glycine-regulatory region of GCV1 and to determine whether the glycine response is regulated in the same manner as GCV2, broad unidirectional deletion constructs of the GCVJ gene were made. Hindiii-Kpni (-310 to +96 of GCVJ) and Xhoi-Kpni ( -130 to +96) fragments from the full-length GCVJ-lacZ fusion construct (containing -1026 to +96 of the GCVJ gene; section 3.1.3) were cloned into Yip357R (Myers et al., 1986) and fused to lacZ, and named pRH102 and pRH103, respectively (Figure 3.9). From the deletion analysis, the glycine regulatory region of the GCVJ promoter was localised to sequences between -310 and -130. Interestingly, it can be seen that glycine induction of GCVJ is mediated mainly by activation rather than repression, since the expression of GCVJ was constitutively low upon the loss of the glycine responsive region (RH103; Figure 3.9). The glycine regulatory region of GCVJ contains 4 potential GCN4 sites. In GCV2, there are 2 potential GCN4 sites within the glycine regulatory region (pRHlO and pRH4; Figure 3.8 A). The possible involvement of these sites in the glycine response will be examined in section 3.4.1. The GCV2-deletion constructs used in Figure 3.8 were also used for the localisation of the region responsible for repression by rich nitrogen sources (or YEPD). The results of the analysis are shown in Figure 3.10. When cells were grown in the medium with ammonium as a sole nitrogen source (Dmin), loss of repression occurred between pRHlO and pRH4 (-313 to -267), as discussed previously. A similar pattern of 92 Regulation ofGCV genes regulation was observed when cells were grown in PROmin (proline as a sole nitrogen source). GCN4 TATA /acZ -1026 c::::« • • • • I I I IRHIOl -263 -180 -162 -143 -88 -310 c======-=====:.:::::::.c:::JII:::::==:::IJ======:::::r:==:J·I • • • • I I I RH I 02 -130 c:::cc==::::Jc::::J RH 1m .:: E '2n D Dmin ;::,E 800 § D Dmin + I 0 mM glycine 5 400 RHIOI RHI02 RHl03 Figure 3.9. Deletion analysis of the GCV 1 promoter to localise sequences involved in the glycine response. The diagram above shows the extent of the deletions with the location (relative to the start codon of CCVI gene) of potential GCN4 sites and TATA box as indicated. Yeast strain Y3 transformed with each of these deletion constructs as single integrated copy were grown in Dmin with or without glycine and assayed forB-galactosidase activity as described in the legend to Figure 3.3B. However, the repression of GCV2 expression m a good nitrogen source (GLNmin) appeared to be due to sequences between -227 and -205 (pRHS and pRH3), and loss of this region derepressed GCV2 expression 15- to 18-fold compared to the level of the wild-type sequence (pRH I). This pattern of regulation was also observed in cells grown in a rich complete medium (YEPD; M. Piper, UNSW, pers. comm.), further supporting that the repression in YEPD is caused by rich nitrogen sources and repression of GCV2 in GLNmin and YEPD is mediated by the same regulatory mechanism (Figure 93 Regulation oJGCV genes 3.7). There are no known regulatory elements in the region between -227 and -205, however, very close to the 3' of this region is a potential GATA site (at -202), which is known to be a binding site for nitrogen-regulatory factors (section 1.4.3). There was also some minor derepression (two- to threefold) when sequences between -313 and -289 (pRHIO and pRHll) was lost. It is not clear whether this is simply due to the loss of the glycine regulatory region, thus relieving some repression of the native GCV2 gene, or there are some interactions between the rich nitrogen source regulatory (YEPD regulatory) element and the glycine regulatory element. Expression of the GCV2 gene in the medium with glycine as a sole nitrogen source (GLYmin) was similarly high regardless of any deletion to at least -190. It appears that the repression by the two regulatory elements is completely relieved under this growth condition. 94 Regulation ofGCV genes -351 fiJ hi I I lxt/1 I :I I. I lSI • RH1 -313 I I I I ll J I I I I Nl • RHlO -289 I ·] j.:;:=:E:1 I I II &4 • RHll -267 I I. I I I I I I lSI • RH4 I Nl 0 GCN4 -227 • RH5 lSI GATA -205 1-'i • RH3 TATABox • -194 I • RH6 2000 1800 ~ 1600 ~ ~ 1400 ~ "a 1200 '--"~ :s 1000 :a 800 - ~ 600 400 GLYmin lIV) 200 RH3 RH6 Figure 3 . 10. Deletio n analysis of the G CV2 p romoter to localise sequen ces involved in the nitrogen source r egulation. Various deletion constructs were transformed into strain Y3 as single-integrated copies and cells grown in media containing different nitrogen sources and assayed for 6-galactosidase activity as described in section 2.9. The diagram above shows the extent of the deletions with the location of potential GCN4 sites, the GATA element and the TAT A box as indicated. Errors were less than 25%. Regulation ofGCV genes 3.2.2. Delimination of GRR (glycine regulatory region) in GCV2 It was shown that the 5'-unidirectional deletion to -267 completely abolished the glycine response of the GCV2 gene. Interestingly, there was a partial but significant loss of this response on deletion of sequences to -289 (Figure 3.8). It appears, therefore, that there are two regions responsible for the glycine response of the GCV2 gene. To determine whether this partial (twofold) relief of repression was due to artefacts of the experiment (eg., bringing activation element from the vector sequence closer to the promoters as deletion of the 5' region of GCV2 proceeded) various internal deletions between -313 and -267 were made. Window-deletion constructs pRH12 and pRH14 were made from pRH4 with pRH7 and pRH8 (section 3.2.1) respectively. After cutting pRH7 and pRH8 with Kpnl, the termini were blunt-ended by T4-DNA polymerase (section 2.3.2). pRH4 was first cut with £caRl and also blunt-ended using Klenow fragment (section 2.3.2), then a 645 bp-GCV2 fragment from pRH4 and fragments of pRH7 (containing -351 to -313 of GCV2) and pRH8 (containing -351 to -289 of GCV2) were isolated after cutting with Xbal. Subsequent blunt-end ligation created pRH12 and pRH14 (Figure 3.11). The resulting plasmids pRH 12 and pRH 14 are identical to pRH I except that they contain deletions between -313 to -267 and -289 to -267 respectively. pRH9 (section 3.2.1) was used to make another window deletion construct (pRH13) by cutting with Kpnl then religating the larger fragment. This deleted the sequence between two potential Gcn4p binding sites ( -310 and -289) of GCV2. Figure 3.12 shows the results of assays using the deletion constmcts, which were consistent with the results from the unidirectional deletion analysis. Loss of sequences either between -310 to -267 or -289 to -267 of the GCV2 promoter region resulted in the elimination of the glycine response, whereas loss of the sequence between -310 and -289 rendered a partial derepression (two-fold) of expression in the absence of glycine in the medium (basal level expression). In conclusion, sequences between -289 and -267 are absolutely required for the glycine response of the GCV2 gene, and there is an additional requirement for the sequences between -313 and -289 for a full-glycine response. 96 Regulation ofGCV genes Am R pRH7 pRH4 or pRH8 Kpnl(-312or-291) i) cut with EcoRI i) cut with Kpnl ii) fill-in 3'-recessed end ii) removal of 3'-overhanging end iii) cut with Xbai iii) cut with Xbal • •Am R Blunt-ended EcoRI (-267)--. r .;, ori pRH7 "(CV2 Blunt-ended or pRH8 Xbal (+378)- Kpnl (-312 or -291) L Ligation Figure 3.11. Construction of internal deletions of the GCV2 promoter region. pRH4 and either of pRH7 or pRHS were used for the construction of pRH12 and pRH14. pRH7 and pRH8 contain a unique Kpnl site (mutated from a potential GCN4 site) at -312 and -291 respectively (section 3.2.1). Consequently, the resulting plasmids had deleted GCV2 promoter regions of -312 to -267 (pRH12) and -291 to -267 (pRHl4) from the pRHl construct. To further dissect the glycine-regulatory region of GCV2, different overlapping fragments of the region between -291 and -266 which delimits the absolute requirement for the glycine response, were inserted in both forward and reverse orientations at the 5'- end of the truncated GCV2-lacZ in pRH4. pRH4 contains the largest gross deletion of the GCV2 promoter lacking glycine-specific control. Five sets of double-stranded oligonucleotides designed to have EcoRI overhangs were prepared from pairs of single- stranded synthetic oligonucleotides (section 2.3.4). After ligation of the DNA fragments 97 Regulation ofGCV genes into pRH4, the resulting constructs were sequenced to determine the orientation of each insertion. -gly +gly (fold) -351 I I I I I I I I I I I I I I Si • RHI 204 ± 47 734 ± 194 (3.60) -351 RHI2 540 ± 66 611 ±52 (1.13) -313 -267 -351 I t------1 I I I I I I I I I w RHI3 398 ± 71 880 ± 180 (2.21) -310 -289 • -351 I I I 1-----1 I I I I I I I M RHI4 629 ± 114 686 ± 175 (1.09) -289 -267 • -313 I I I I I I I I I I I I ij • RHIO 2!3 ± 62 625 ± 118 (2.93) 0 GCN4 -289 I I I I I I I I I I N RHII 450 =76 989 ± 246 (2.20) Ill! GATA • • TATABox -267 I I I I I I I I AA • RH4 1254± 350 1231 ± 189 (0.98) Figure 3.12. Window deletion analysis of the GCV2 promoter region for glycine response. On the left is a diagrammatic representation of the window deletion constructs together with some unidirectional deletion constructs. The single line in the window deletion constructs (pRH12 to 14) represents sequences deleted from pRH I. The location of potential GCN4 sites, the GATA element and the TATA box are as indicated. Strain Y3 transformed with each construct was grown and assayed for 13- galactosidase activity as described in the legend to Figure 3.3B. Fold-induction of expression from Dmin (-gly) to Dmin supplemented with 10 mM glycine (+gly) is indicated in brackets. One of those pRH4-derived constructs, which had an insert of the II bp- sequence, 5'-TGACTCTTCTT -3' (fragment 4; figure 3.13), restored the glycine response of GCV2 in both forward and reverse orientations. However, this fragment did not completely repress GCV2 expression, rather it showed a two-fold effect compared with the wild-type (pRH I) which showed an approximate three- to fourfold repression. This is comparable with the results from deletion analyses since there is an additional requirement in the region between -310 and -289 for the full glycine response while the region between -289 and -267 was absolutely required. When the sequence between -289 and -267 (pRH13; Figure 3.12) was lost, there was a twofold elevation in the basal level expression. Therefore, it is considered that fragment 4 contains the sequence that is absolutely required for the repression. 98 Regulation ofGCV genes This fragment contains the sequence 5'-TGACTC-3' which is known to be a potential binding site for Gcn4p and Bas lp transcription factors (section 1.5.3) as well as an additional 5'-CTTCTT -3' motif. Fragment 5 also contains a possible GCN4 site (5' AGACTC-3'; Figure 3.13), but the insertion of this fragment did not confer a glycine response. The 11-bp sequence (5'-TGACTCTTCTT -3') which participates in the glycine response of CCV2 was used to search the other known glycine responsive genes, CCVI and CCV3. The promoter of CCVI shares remarkably strong homology with CCV2 over about 50 bp including the 5'-CTTCTT -3' motif (at -177, relative to the start codon) as shown in the alignment in Figure 3.13B. From this alignment, it can be deduced that the most probable glycine response element is 5'-CATCN?CTTCTT-3'. CCV3 showed less obvious homology to either CCVI or CCV2 in the promoter region, although there is a region of conservation of sequence across all three genes; in this region of CCV3 the CIT triplet occurs three times, with two adjacent. The CCV3 gene showed the least prominent glycine response among the CCV genes (Figure 3.5), and this may be a reflection that this gene does not contain an optimal glycine-responsive element. It was shown that expression of the CCV2 gene is also regulated by L-methionine (Hong eta!., 1999). Interestingly, the region responsible for this L-methionine regulation coincided with the glycine-regulatory region of CCV2 and a CCVl-lacZ fusion construct was also found to respond to L-methionine, whereas a CCV3 -lacZ construct did not when transformed in the same strain (M. Piper, UNSW. pers. comm.). This supports the idea that the detailed or fine regulation of the CCV3 gene is different from that of the other CCV genes, and this may be due to the fact that CCV3 contains a less than optimal glycine response element. In order to further define the important bases in the 11 bp sequence from CCV2 (fragment 4: 5'-TGACTCTTCTT-3') which restored the glycine response, and also to examine the contribution of some potential elements within the region between -310 to -289 (for full glycine response), site-directed mutagenesis studies were performed (Figure 3.14). 99 Regulation ofGCV genes -291 -266 I I I 5'-TG ACT c:rT C TT:CAGGT1 TA qA C T C CT-3' CDI I @ I ® @ I ®· A. Dmin+ or icntation Dmin !OmM glycine forward 880 1045 l reverse 885 1253 forward 983 ll65 2 reverse 979 1113 forward 918 981 3 reverse 1193 1073 forward 497 1018 4 reverse 484 1044 forward 1061 1347 5 reverse 1010 1112 B. * * * * * **** * * ****** ** * * * *** * **** * ****** * GCVl GATA'TCACCTATTTTGTTCATCGCCGTGACTTCTTTCGGCA-GGGCGACTCTGCTCAGGAGCCCTGACTCGTGTTG GCV2 GGTTGAATCGTTACCCGACATCTCTGACTCTTCTTGAGGTATAGACTCCTCCTTTTCGGAGTCATGACTCTCAGAG GCV3 ACTGCTTGTGTAAGCCTTCATCTTCTTG-CTTTAGCTGATGCAGCAGGTCGCGAA-CAGTGTCCTGCACTGGGGGT * ** * ** ***** ** * * * ** * * * *** ** * Figure 3.13. Search for the sequence responsible for the glycine response. A. Sequence between -291 and -266 of CCV2 that can restore the glycine response to the truncated CCV2 promoter in pRH4 were sought by DNA fragment insertion. Fragments 1 to 5 indicated in the diagram were cloned in both forward and reverse orientations at the 5' -boundary of the GCV2 sequence in pRH4. Cells containing each construct was grown and assayed for H-galactosidase activity as described in the legend to Figure 3.3B; all errors were less than 26o/c. B. Comparison of the promoter regions of the glycine-responsive genes (CCVI: -206 to -132: CCV2: -315 to -240; CCV3: -314 to -241). Asterisks indicate identity to the central CCV2 sequence of either CCVI or CCV2, not homology between all three. 100 Regulation ofGCV genes The mutations that were made involved alteration of 4 bp in the core 5' TGACTC-3' consensus of the GCN4 sites, and mutations which altered the 5'-CTTCTT- 3' or 5'-ACATCT -3' hexanucleotide sequences to 5'-GGT ACC-3'. Mutations of each 5'-CTT -3' motif within the CTTCTT sequence to 5'-AGG-3' were also made separately for the analysis (section 2.3.6). Mutation of the GCN4 sites separately or in combination did not make significant differences to the level of basal expression or to the glycine response (pRH7, 8, and 9; Figure 3.14). Together with the results of the DNA fragment insertion study (Figure 3.13A; fragment 5), it appears that none of the three potential GCN4 sites within the glycine responsive region (-313 to -267) are responsible, at least, for the glycine response of the GCV2 gene. However, change to the 5'-CTTCTT-3' sequence led to a more than two-fold increase in the basal level expression, reflecting a partial loss of glycine response (pRH16). When this mutation was combined with mutation of an adjacent GCN4 site (pRH19), there was no additional loss of glycine response, confirming that the GCN4 site of G CV2 does not play an important role in the glycine response. Maximal derepression was observed when the 5'-CATC-3' sequence was mutated in conjunction with the CTTCTT sequence (pRH20). In this construct, the basal level of expression was elevated about 3-fold, and most of the glycine response was lost. These results support that the CATCN7CTTCTT sequence is the glycine response element as was deduced by GCVI and GCV2 promoter alignment (Figure 3.13B). Interestingly, partial mutations within the CTTCTT sequence showed no significant effect (pRH17 and pRH18), which may provide an insight into the nature of the cognate binding protein which recognises this element. In the GCV3 gene, no "optimal" glycine response element (CATCN7CTTCTT) was found, instead, there are multiple CTT sequences found in the promoter region (thirty 5'-CTT -3' sequences between -500 and the start codon). Some of these sequences might provide sub-optimal binding sites for the binding factor, and the glycine response may occur by the additive effect of these bindings. 101 Regulation ofGCV genes GCN4 GATA TATA /""~' Dmin+ CCCGGTkGAATCprrACCC~AGG Dmin -318 -277 lOmMGly (fold) ------1~--cr:=c=Jf--- RHl 204 ± 47 734 ± 194 (3.60) RH7 189 ± 47 663 ± 62 (3.51) RH8 289 ± 52 743 ± 76 (2.57) RH9 270 ± 47 805 ± 57 (2.98) RHl7 243 ± 18 797 ± 130 (3.28) ------1~--cr=DC> Figure 3.14. Site-directed mutagenesis of the GCV2 promoter to localise sequences involved in the glycine response. On the left is shown the location of potential GCN4 sites (dotted boxes), the GATA clement (shaded box) and the TATA box (filled box). Crosses represent mutated motifs for the putative GCN4s and CATCN7CTTCTT elements. Relevant sequences between -318 and -227 of GCV2 were mutated separately or together in the full length pRHI construct, as indicated in the diagram. The effects of each mutation on expression was assessed by measuring B galactosidase specific activity(± standard deviation) as described in the legend to Figure 3.3B. 102 Regulation ofGCV genes 3.3. The glycine regulatory region (GRR) in heterologous promoters 3.3.1 The glycine response of GCVJ and GCV2 is mediated by repression In order to test whether the glycine regulatory region (GRR) determined by the above analyses (section 3.2) has activity in a different sequence context, the 42 bp fragment (-309 to -267) of GCV2 spanning the important core and flanking sequences was inserted into the Xhol site located between the UASc and TATA box elements of a complete CYCJ promoter fused to a lacZ reporter gene in plasmid pLGL'l-312S (section 2.1.5). The GCV2 fragment -322 to -248 was first amplified by PCR using primers harbouring X hoi restriction sites (31 OF and 260R; section 2.1.4) and the internal 42 bp Xhol-fragment was isolated and cloned into the pLGL'l-312S. The orientation and number of insertions were confirmed by sequencing. Two constructs were tested; one contained an insert of one 42 bp fragment, the other carried two inserts in a tandem duplication (Figure 3.15 B). The native CYCJ promoter produced a very high level of expression (a more highly expressed promoter than that of GCV2), regardless of the presence or absence of glycine in the medium. Insertion of one element into the CYCJ promoter led to a two-fold repression of expression in medium lacking glycine, and much of this repression was relieved by addition of glycine to the medium. When two 42 bp fragments were inserted, the extent of repression was greater than five-fold, and addition of glycine caused a fourfold increase to about 75% of the level seen in the intact CYCJ promoter. While overall reduced expression in derepressing conditions might be due to changes in promoter spacing on insertion of the heterologous sequences, these results demonstrate clearly that the elements confer a glycine response in an heterologous system, and the GRR from GCV2 mediates the glycine response by repression. 103 Figure 3.15. The glycine-regulatory regions (GRR) from CCV2 and CCVI confer repression on an heterologous promoter that can be relieved by glycine. A. The arrangement of elements in intact VASe containing plasmid pLGLl.-312S (section 2.1.5) including the location of the Xhol restriction sites used for insertion of the glycine response region. B. The 42 bp CCV2 glycine regulatory region (GRR) from -309 to -267 was inserted as either one copy (forward orientation (F)) or two tandem copies (reverse orientation(R)) in to the Xhol site of the CYCI-lacZ promoter of pLGLl.-312S. C. The 31 bp sequence from the CCVI spanning -193 to -162 confers a glycine response on the VASe-containing heterologous CYCJ-lacZ promoters. The GRR of CCV I was inserted as either one or two copies (in both forward (F) and reverse (R) orientations) into the CYCI-lacZ promoter of pLGLl.-312S. Each construct was transformed in strain Y3 and assayed forB galactosidase in triplicate in cells grown on Dmin with and without the addition of 10 mM glycine as described in the legend to the Figure 3.3B. Regulation ofGCV genes A. pLG~-3!2S Xhol-Sall-Xhol Smal(-312) (-178) UASC [H CYCJj lacZ j -275.-225 B. 0Dmin 0 Dmin + 10 mM glycine ~ '-= g 2000 c. rJl 0 copy 1 copy 2 copies Copy number c. 9000-,------. 6000 3000 0 copy I copy (F) I copy (R) 2 copies (F) 2 copies (R) Copy number 104 Regulation ofGCV genes A putative GRR of the CCV I was revealed by promoter sequence alignments with the functional GRR of the CCV2 gene (Figure 3.13B). The region from -193 to -162 of CCVI showing strong homology with the GRR of CCV2 also contains the 5' CATCN?CTTCTT-3' element. This putative GRR was inserted into the Xhol site of the CYCI-IacZ reporter plasmid pLG~-312S described above to test its functionality as a repressor-binding element. The CCVI fragment -193 to -162 with Xhol cohesive ends was produced by annealing a pair of oligonucleotides (TgrrF and TgrrR; section 2.1.4). The result of the analysis is shown in Figure 3.15 C. The CCVI fragment acted as a repressor with a greater effect of more copies, and it also retained glycine responsiveness, which closely resembles the situation seen with CCV2. This result is further supported by an insertion construct where the CCVI glycine response region was cloned in front of the truncated CCV2 promoter in pRH4 which is the largest CCV2 promoter deletion lacking glycine response (M. Piper, UNSW, pers. comm.). This restored a glycine response to the CCV2 construct with the CCVI element acting in a similar way to the CCV2 element. In conclusion, the GRR of both CCV2 and CCV1 genes is functional in an heterologous promoter and the glycine response in this heterologous systems is mediated by the presence of glycine acting to relieve repression. 3.3.2 The glycine response can also be mediated by activation Deletion analysis of the CCVI upstream sequence gave the interesting result that unlike the negative control seen for CCV2, the deletion of sequences (between -310 and -130) led to a loss of glycine inducibility of expression (Figure 3.9). Therefore, the glycine response of the CCVI gene is mediated by activation rather than repression at least in its native context. This result is the opposite of that seen in section 3.3.1, showing the GRR of CCV I mediates repression. To investigate this interesting point, -193 to -162 of CCVI was inserted into the blind (UAS-less) CYCI-lacZ reporter plasmid pLG~-312SS at the Xhol site. The plasmid pLG~-312SS is identical to pLG~- 312S except that the UAS sites in the promoter region of the CYCJ gene were removed (section 2.1.5). 105 Regulation ofGCV genes 50 6ooo.------, .S -;;,E 40 :§ 0 I 30 -'=' :~ 20 < T "u t;: ·c u 10 c. 'l; 0 0 copy 1 copy (F) I copy (R) 0 copy 4 copies (R) Copy number Copy number Figure 3.16. The glycine regulatory region from GCV 1 confers a glycine response on UAS-less CYCJ-/acZ promoters via activation. The 31 bp GCV I promoter region from -193 to -162 was inserted as either one copy (forward (F) and reverse (R) orientation) or four copies (reverse orientation) into the X hal site of the blind (UASc-less) CYCI·lacZ promoter in pLGt--312SS (section 2.1.5). Each construct was transformed into Y3 and assayed forB-galactosidase activity. The results shown in Figure 3.16 were virtually the opposite of those seen in the UASc-containing constructs (Figure 3.15C). One copy caused strong activation of the reporter even without the addition of glycine to the medium, with the addition of glycine to the medium causing a modest induction. In another construct in which four copies of the construct were present in tandem array there was substantial activation of expression of the reporter in the absence of glycine, and a greater than 14-fold further induction on the addition of glycine. This result reflects the situation in the native gene where GCV1 is positively regulated and also indicates that the GRR may function as either an activator or a repressor depending on its context, but that the response to glycine is retained. This has interesting implications for the way these cis-acting elements function. In section 3.5, the examples of gene regulation exerted by transcription factors that can act as both a repressor and an activator will be discussed to aid in the understanding of the mechanism that may govern the regulation of the GCV genes. From deletion analysis of the GCV2 promoter (section 3.2), it was shown that there appear to be two regions responsible for glycine response, one is absolutely 106 Regulation ofGCV genes required and the other is required for the full glycine response. It is possible that there are multiple transcription factors bound to these regions to mediate the regulation and the interaction of these factors may confer an activation or a repression of transcription depending on the physiological state of the cell. In addition, there are several potential GCN4!BAS I sites within the GRR of GCVJ and GCV2. Given the role of the GDC in one-carbon metabolism and Gcn4p and Baslp in amino acid synthesis and purine synthesis, the involvement of GCN4/BAS I in the regulation of GCV genes seems quite plausible. In section 3.4.1, it will be examined further whether these activators are in any way involved in the GCV gene regulation. It was also shown that the expression of the G CV2 gene is repressed in the presence of rich nitrogen sources (or in YEPD), which is mediated by the promoter region between -227 and -205. Very close to this region (at -202) is the 5'-GATAA-3' sequence which is known as the binding site for the NCR-regulatory factors. In section 3.4.2, using mutants that are devoid of the nitrogen-regulatory factor(s), it will be investigated if any of these affects the regulation of the GCV2. 3.4. Expression study of the GCV genes in various mutants 3.4.1 gcn4 and bas I mutants There are multiple potential binding sites for the transcriptional activator, Gcn4p (5'-TGACTC-3') in the upstream region of the GCV genes. The GCV2 gene contains seven potential sites, the GCV I gene has four sites and GCV3 contains three sites in their upstream regions (Figure 3.2). These are also known potential sites for the Baslp transcription factor (section 1.5.3). Interestingly, these sites are not only abundant in the promoter regions of the GCV genes, but also found within the glycine-regulatory region (GRR) of the GCV2 and GCVJ genes (section 3.2). Since the GDC can participate in the synthesis of glycine by its reversible reaction, and Gcn4p plays an important role in the regulation of genes involved in amino acid biosynthesis, it is the aim of this section to 107 Regulation ofGCV genes investigate whether there is any role of these potential cis-acting elements in the regulation of the GCV genes. Transcriptional activation by DNA-binding proteins is often synergistic in that the effect of multiple recognition elements for an individual activator in a single promoter is greater than the sum of the effects of the single recognition elements (Ptashne, 1988). This is even observed when the binding sites are recognised by distinct proteins and the effect can be explained by cooperative binding of the proteins to their sites in the promoter. Besides the possibility of direct or indirect physical interactions between the regulators, synergism can in principle also be achieved kinetically when the activators stimulate different steps in the transcription initiation pathway (Herschlag and Johnson, 1993). Promiscuous synergism between regulators is a fundamental aspect of eukaryotic transcription and constitutes an important basis for the extraordinarily diverse patterns of gene expression mediated by upstream elements. A B 1500 1200 D Dmin .:: c E Dmin+ E D I 0 mM glycine E "" 1000 ""~ 800 "'~ E 5 5 .2 Q > ·:;: u u ~ 500 ~ 400 u ~ ·-·u "u u c. c. u: r/)" 0 0 GCN4 gcn4 gcn2 GCN4 gcn4 gcn2 Figure 3.17. Expression of G C V 2 (A) and G C VI (B) in mutants affected in the general amino acid control. The /acZ-fusion constructs of GCV2 (pRH2) and CCV I (pRHI02) which contain all the potential GCN4 sites were transformed separately in to different yeast strains. The GCN4 strain is the wild-type strain which has intact GCN4 and GCN2 genes (F 113; section 2.1.6). The gcn4 and gcn2 strains are single mutants of GCN4 and GCN2 respectively but arc otherwise isogenic to the GCN4 strain. Cells were grown and assayed for B-galactosidase activity as described in the legend to Figure 3.3B. 108 Regulation of GCV genes To investigate the role of the multiple GCN4 sites, the gcn4 and gcn2 mutants were employed that are devoid of the transcriptional activator, Gcn4p and the protein kinase, Gcn2p respectively. These strains were shown to be impaired in enzyme derepression under conditions of amino acid starvation (Gcn- phenotype) since Gcn4p and Gcn2p are direct or indirect positive regulators in general amino acid control (section 1.5.3). The expression patterns of GCV2 and GCVJ genes in these mutants were found to be very similar (Figure 3.17). The glycine response was retained in all mutants tested, although the expression levels were lower in the gcn4 mutant. The reason for this difference is not clear, however, a possible explanation is that Gcn2p is an indirect activator for the general amino acid control and acting as a sensor for the cellular nutritional condition. In other words, it is further away from the control system. Gcn4p is a direct activator of many genes involved in amino acid biosynthesis, and deletion of GCN4 probably affects many aspects of cellular metabolism. It appears therefore, the reduction of the expression levels of the GCV2 and GCVI genes in the gcn4 mutant may reflect indirect effects resulting from the gcn4 deletion disturbing other cellular processes. A similar reduction of expression levels in a gcn4 mutant compared to the wild-type strain has also been observed in the study of the GCV3 gene (Nagaraj an and Storms, 1997). Another transcriptional activator protein, Bas I p is also known to bind 5' TGACTC-3' and is involved in the regulation of genes for purine and histidine biosynthesis (section 1.5.3). Since the GDC plays an important role in one-carbon metabolism, the expression of the GCV2 gene was tested in mutants lacking Bas 1p (Figure 3.18). The basi mutants were derived from the GCN4 wild type or gcn4 mutant strains by the gene replacement technique described in section 2.5.3 using the BASI deletion plasmid, pB 1559 (section 2.1.5). Again, as in mutants in affected in the general amino acid control system (Figure 3.17), all strains with the basi deletion retained their glycine response. Therefore, Bas 1p is also not directly involved in the glycine response of the GCV2 gene. However, deletion of the BAS I gene caused a slight, although not significant, reduction (about 1.5 109 Regulation ofGCV genes times) in the expression of the GCV2 gene. This overall reduction in GCV2 expression is observed in both basi mutants tested (GCN4/basl and gcn4/basl relative to the GCN4/BASJ and gcn4/basl strains respectively). It is not clear whether this reduction in the expression level is indirect due to the loss of Bas 1p which may affect the expression of many genes involved in purine and histidine biosynthesis. 1500,..------, D Dmin D Dmin + 10 mM glycine c E -eo :§ 1000 Ec .2 ~ tl < .!< 500 GCN4/BAS I GCN4/basl gcn4/basl Figure 3.18. Expression of GCV2 in mutants of the general amino acid control. The GCV2-lacZ fusion construct (pRH2) was transformed separately into different isogenic strains. The GCN4 strain (FII3; section 2.1.6) is the wild type strain which has the GCN4 and BASI genes intact. The GCN4/basl is the single null-mutant of the BAS I and gcn4/basl strain is the double mutant of both GCN4 and BASI. Cells were grown and assayed forB-galactosidase activity as described in the legend to Figure 3.3B. However, a similar result was reported in a study of BASI control of the GCV1 gene expression in de novo purine synthesis. Analysis of the genes, GLNJ, SHM2 and MTDJ which are required for synthesis of glutamine, glycine and 10-CHO-THF during de novo purine synthesis showed that their expression is repressed by adenine and requires the transcription factors Baslp and Bas2p (Daignan-Fornier and Fink, 1992; Denis and Daignan-Fornier, 1998). Repression of GLNJ expression by adenine is not unexpected since it has been estimated that 50% of glutamine produced by the cell is used for purine biosynthesis (Magasanik, 1992). Mtdl p possibly plays an important role in 110 Regulation ofGCV genes supplying 10-CHO-THF to the purine pathway, since it catalyses the synthesis of 10- CHO-THF only in the oxidative direction (West et aL, 1996). Interestingly, in the case of CCVi, although there was no repression by adenine in isogenic wild-type, basi and bas2 strains, the expression level was decreased two-fold in a basi mutant compared to the wild-type strain, which indicates that there may be some role for Baslp in the regulation of CCVI (Denis and Daignan-Fornier, 1998). It is known that at least 25% of purine synthesis is contributed to by the mitochondrial one-carbon metabolism pathway (Pasternack et aL, 1994a). Furthermore, the expression level of the SHM2 gene was much higher in a bas2 mutant than in a basi mutant strain (Denis and Daignan-Fornier, 1998). Since all genes activated by BASi and BAS2 were equally affected by mutations at either of these loci (Daignan-Fornier and Fink, 1992; Rolfes eta/., 1997; Tice-Baldwin et aL, 1989), and a LexA-Baslp fusion is unable to activate transcription in the absence of Bas2p (Zhang et a/,, 1997), this suggests that Bas I p can interact with some other factors to activate transcription in the absence of Bas2p on the SHM2 promoter. Similar regulation of the ADE3 gene was observed by Northern analysis and two-dimensional protein gel analysis (Denis eta!., 1998). From the results of mutants study shown in Figure 3.17 and 3.18, it is apparent that Gcn4p and Baslp are not directly connected to the "glycine response" of CCV genes, which is consistent with the results of the site-directed mutagenesis on the potential GCN4/BAS 1 site within the GRR showing no significant affect on the glycine response of the CCV2 gene (Figure 3.14). However, both gcn4 or basi deletion led to a reduction of the overall expression level. This may suggest that these sites are involved in setting up the full activation of the genes. These results also cannot exclude the possibility that the sites are functional in other specific cellular conditions. This is supported by the observation that Baslp may have some role in the regulation of CCV genes. Composites of multiple binding sites for different regulatory factors are often responsible for the specific regulatory properties of a promoter (Miner and Yamamoto, 1991). Moreover, many purified DNA-binding proteins bind with equal affinity to sites that share only minimal nucleotide sequence similarity, and a given cis-regulatory Ill Regulation of GCV genes sequence can frequently be the target of more than one DNA-binding protein (Struhl, 1993). This indicates that multiple protein-protein interactions that are highly specific for the given upstream element can be ultimately responsible for the appropriate regulation of a target gene. The possibility of such an interplay between regulatory factors increases the precision as well as the flexibility of transcriptional regulation. Certain nutritional conditions and/or the use of one-carbon metabolic mutants in which glycine synthesis or purine synthesis is forced via the GDC can be tested for this hypothesis. 3.4.2 NCR-regulatory mutants. Yeast cells in the natural environment probably require numerous different regulatory systems to operate simultaneously, and with a magnitude of response appropriate to suit environmental conditions. Glycine can supply nitrogen to the cells via the GDC and it was shown that the GCV genes are regulated by nitrogen sources (section 3.1.3) and the cis-acting repressor element was localised between -227 and -205 of the GCV2 promoter (section 3.2.2). Immediately 3' of this region is a potential GAT A site (at -202), which is known to be a binding site for nitrogen-regulatory factors (section 1.4.3). It is the aim of this section to determine whether regulation of the GCV2 gene by nitrogen sources is mediated by this potential GAT A site. For this, different isogenic mutants of nitrogen regulators were used. These included deletion mutants of the four known GATA factors (Gln3p, Uga43p, Nillp and Gzf3p), either as single mutants or as double mutants with different combinations of regulators. Briet1y, Gln3p and Nil! pare activators and the Uga43p and Gzf3p are repressors of the NCR-dependent genes (section 1.4.3). Each mutant strain was transformed with pRH4 (Figure 3.8) as a single copy integrant. This construct was chosen to eliminate the possible effects of the glycine regulatory region (GRR). Transformants were grown under five different conditions: YEPD, GLNmin, PROmin, GL Ymin and Dmin. If any of the GATA factors are involved in the repression of GCV2 gene, it was expected that the expression of the GCV2 gene would be derepressed in some mutant(s) under rich nitrogen conditions. Therefore, cells were grown either in complex medium 112 Regulation ofGCV genes (YEPD; Figure 3.19 A) or in medium containing L-glutamine as sole nitrogen source (GLNmin; Figure 3.19 B). From the results, it is clear that the repressors of the NCR system did not significantly affect the expression of GCV2 gene. However, there was two- to threefold derepression in strains deleted for the activators of GATA factors. This expression level was further derepressed when both of the activators were deleted (five- to six-fold relative to the wild type strain). A. B. 150...,------,l"' 400,------, = T ~ _§ 100 0 1 + E 5 c > u 50 < u !..:: ~ ~. ':1 " ~ "'~ ~ ....,~ ~ "'~ "'~ "'"" Figure 3.19. GCV2-lacZ expression in different nitrogen regulatory mutants. Each strain was transformed with pRH4 (Figure 3.8) and grown either in D-glucose complex medium (YEPD; A) or in a minimal medium with glutamine as sole nitrogen source (GLNmin; B). B-galactosidase assay were as described in the legend to Figure 3.3A. It is interesting and curious that derepression of the GCV2 gene in both nitrogen rich conditions was observed when the activators were absent. This may be due to the complexity of the nitrogen regulatory network (Figure 1.4 ), in that the loss of activators caused the reduced expression of repressors. However, the loss of either repressor did not significantly change the level of expression. It appears, therefore, the repression of GCV2 under rich nitrogen conditions is not directly caused by the GATA factors, but rather results from indirect effects. 113 Regulation (d. GCV genes PROmin c. 600,------. c E T I -""E 400 I I -0 []! E 5 1 1 ·;;~ T/.' T 200 -<" t ·c;'-<=" 0. (11" 0 ' ' .,. ' '"::l ' '"::l ::: ~ ""~ ·~ ~ "'~ ""~ .,.""' " ""' D. Dmin Dmin 900 .,------, 100.------. c c T E ~ I en 75 r ..§ 600- T T .§ f 0 0 E 7= E " I T 5 so Tr ,q T ,q .~ $ r:§ T 1 ·~ -< 300 -<" " u ;..::" ir:::::: :.= 25 ·g u 0. 0. (11 (11 0 ' ' '- .,. '"::l ::: ';:;, ;::: '- .,. '"::l' ""' .s""' ·~ "' ""' .s""' ~ -<: -"' " ~. " ~ "' "' .,.~"' "' "' '"'~ '"' .,. ~ ~ "" '< " " """ Figure 3.19. G C V 2 -lac Z expression in different nitrogen regulatory mutants. Each strain was transfonned with pRH4 or pRH2 and grown either in minimal medium containing proline as a sole nitrogen source (C) or in minimal medium with ammonium as sole nitrogen source (D). B galactosidase assays were as described in the legend to Figure 3.3 A. Transformants of pRH4 are used in figure C and the left panel of figure D. On the right of figure D shows the results of the pRH2 trans formants. The loss of both activators probably severely disturbs nitrogen assimilation in the cell because repressors of the NCR system are also regulated by activators (Figure 1.4) and it was shown that at least one positive GATA factors is required for the negative GATA factors to be expressed (Soussi-Boudekou et al., 1997). Consequently, in cells 114 Regulation ofGCV genes devoid of both activators of the NCR system, the control of nitrogen catabolic genes will function abnormally. In this case, cells might sense the abnormality in the nitrogen assimilation system, and require the GDC for utilization of glycine that would not be normally happening and thus derepresion of the GCV2 gene occurs. It is also interesting that some derepression was observed with the loss of Gln3p but not by the loss ofNillp in cells grown in YEPD and vice versa for GLNmin although both media are considered to be rich in nitrogen. The contribution of activators' action might be different in different rich nitrogen media because of the composition of the media. It is difficult to determine which factor(s) is/are causing this difference because YEPD contains a variety of nutrients such as good/poor nitrogen sources, glycine, and other one-carbon metabolites that possibly affect the regulation of the GCV2 gene. The expression pattern of the GCV2 gene was also examined in other growth conditions including PROmin (medium with proline as sole nitrogen source) and Dmin (ammonium as sole nitrogen source). In cells grown in PROmin and Dmin, the expression patterns were very similar (transformants of pRH4), except that there was a slight elevation of the expression level (1.5-fold) in a gln3 mutant in Dmin grown cells. Interestingly, this slight elevation was reversed when pRH2 (the GCV2-lacZ construct containing the intact GRR; Figure 3.8) transformants were tested. This implies that there is a possible interaction between the nitrogen-regulatory element and the GRR of the GCV2 gene (Figure 3.19 C and D). In support of this idea, the GCV2 expression pattern in medium containing glycine as sole nitrogen source showed a similar reversal effect when comparing the expression levels of the GRR-less with the ORR-containing GCV2-lacZ constructs (Figure 3.19 E), although the effect of the gln3 deletion was opposite to that of Dmin grown cells. In all the conditions tested for GCV2 expression, it is clear that the negative regulators of the NCR system had little, if any, effect on the control of the GCV2 gene. This is reasonable considering Uga43p and probably Gzf3p require two GATAA containing sequences oriented tail to tail or head to tail, 15 to 35 bp apart (Cunningham 115 Regulation ofGCV genes and Cooper, 1993; Cunningham eta/., 1994), and there is only one GATAA element in the GCV2 upstream region. E. 600 1600 " ? E I T ] Ol) 1200 -E'"' t E r 400 1 -0 T T T T 0- 7 E r.t E r+-1"'::::: ::::::1'" 5 w 5 ·.·.· ..·.·.·. ~ T ~ 800 -~ > u u .. ..: 200 T ..: u u <= 400 ·u s t ·u<= u u c. c. "' "' ... 0 ' ' ' ' 0 h ":! ":! ;=.;: !"~'"', s "" ~ - "'"" "' ~. '"'~ "" ~ ""'"" .s"" "'"" "' ""~ "' Figure 3.19. GCV2-lacZ expression in different nitrogen regulatory mutants. Each strain was transformed with pRH4 (on the left) or pRH2 (on the right) and grown in minimal medium containing glycine as sole nitrogen source (GL Ymin). B-galactosidase assays were as described in the legend to Figure 3.3A. As shown in Figure 3.19 A and B, the loss of activators altered the level of expression of the GCV2 gene, probably via an indirect effect. Likewise, the reversal effect in the gln3 mutants in the GRR-less and GRR-containing GCV2-lacZ constructs (Figure 3. 19 D and E) may not be due to a direct interaction of Gln3p and GRR-binding protein. It is possible that there exists yet another nitrogen source regulatory protein which regulates the CCV genes under different nutritional conditions which is directly or indirectly related to the four known GATA factors and which may interact with the GRR binding protein. The nitrogen-regulatory network of NCR controlled by the four GAT A factors was shown to be incomplete and it is thought that there are other negative factors involved in nitrogen repression. This has been suggested on the basis of the observation that some nitrogen-pathway genes such as GAP I and UGA4 remain partially sensitive to 116 Regulation ofGCV genes nitrogen repression m cells lacking Ure2p, Gzf3p, and U ga43p, all the negative regulators of NCR known so far (Soussi-Boudekou eta!., 1997). Taken together, the four GAT A factors do not appear to directly regulate GCV expression, and there may be another nitrogen regulatory protein that possibly interacts with the GRR-binding protein to control the GCV genes' expression. The regulation of GCV genes under different nitrogen sources by an as yet unknown regulator is further supported by the absence of the GAT AA elements in the promoter region of the GCV I gene which was also shown to be regulated by different nitrogen sources (Figure 3.2). In the search for a potential DNA element that is responsible for the nitrogen regulation of GCV genes, the region between -227 and -205 of GCV2 (section 3.2.2) was compared with GCVI and GCV3 promoter regions. A very good candidate for the regulatory element is a palindromic sequence, 5'-AAGGACCTT-3' that is located at -211 bp relative to the start codon of the GCV2 gene, adjacent to the 5' of the GATAA element. Very similar sequences were found in the other GCV genes through alignments with this potential element. In GCVJ, 5'-AAGGACCTc-3' (lower case indicates the mismatch from that of GCV2) is located at -382 bp relative to the start codon. In GCV3, the sequence 5'-AAGGACCaT-3' is found at -495 (relative to the start codon) and 5' AgGGACCTc-3' is also found at -123. A preliminary deletion analysis with the GCVJ gene indicated that the regulatory element is probably located between -1026 and -310 (pRHlOl and pRH102; Figure 3.9), which further supports the idea. The functionality of this putative element needs to be examined further by inserting it into heterologous promoters together with detailed promoter analyses (deletion analysis, site-directed mutagenesis) as well as in vitro analyses including the study of DNA-protein interactions and footprinting. This work is beyond the scope of the present thesis in which the glycine response became the major focus of further research, but is continuing in collaboration with M. Piper (PhD student, UNSW). 117 Regulation ofGCV genes 3.5 Conclusion. In this chapter, it was shown that the GCV genes are regulated by glycine and rich nitrogen sources. Promoter analyses of the GCV2 gene revealed that these controls are mediated by two different DNA elements. Promoter sequence alignments of the GCV1 and GCV2 genes, together with detailed genetic analyses showed that the glycine response was mediated by a 5'-CATCN7CTTCTT-3' motif (GRR; glycine regulatory region). It was also shown that sequence immediately 5' of this element has a minor role for the full glycine response of the gene. An interesting finding of this work is that the GRR could mediate the glycine response by both activation and repression: in GCV2, the addition of glycine in the medium relieved repression; in GCVJ, the addition of glycine activated gene expression. The dual-functionality of the GRR of the GCV genes has been confirmed by inserting this region into an heterologous promoter context. Within the GRR of GCV2 gene, there is a Gcn4p/Baslp-binding site. In strains lacking Gcn4p and/or Bas I p, the glycine response of the GCV2 gene was retained but overall expression levels were decreased, indicating that these proteins may have a role in the regulation of the GCV2 gene, but not the glycine response. Further repression of the GCV2 gene when cells were grown in rich medium is mediated by sequence between -227 and -205. Adjacent to this region is the sequence 5' GATAAG-3' which is known to be the binding site for NCR-regulatory proteins. Studies with mutants lacking these regulatory factors showed that they are not directly related to repression of GCV2 under rich nitrogen conditions. A potential DNA regulatory element responsible for this nitrogen regulation was suggested through sequence alignment; a palindromic sequence, 5'-AAGGACCTT-3' which is found in the promoters of all three GCV genes. Since the glycine response could not be conferred by glycine analogues in the medium, it appeared unlikely that glycine is the actual effector molecule signalling the need for the expression of GCV genes. In chapter 4, in vitro approaches using electrophoretic mobility shift assays (EMSA) and footprinting were used for identification of the effector molecule to establish a revealing link between gene expression and one- 118 Regulation ofGCV genes carbon metabolism. Studies of protein-DNA and protein-ligand interactions also gave invaluable information for understanding the mechanism of transcriptional control of the CCV genes. 119 DNA-protein-signalling molecule interactions Chapter 4: DNA-PROTEIN-SIGNALLING MOLECULE INTERACTIONS 4.1 Electrophoretic Mobility Shift Assay (EMSA). 4.1.1 Introduction. A detailed understanding of transcriptional control requires knowledge about the cis-acting sequences that are essential for the expression and regulation of specific genes. It is also important to understand the proteins that interact with these sequences and the molecules that affect the activity of these proteins such that they influence transcription in the appropriate physiological manner. The previous chapter concentrated on the identification and localisation of DNA sequences that are responsible for the regulation of the GCV genes. In this chapter, detailed analyses of interactions between the glycine regulatory region (GRR) and the protein(s) binding to the GRR will be studied to resolve in more detail the nature of this glycine regulation and, to help identify the transcription factor(s) responsible. Identification of a signalling molecule will also be shown and discussed in terms of its physiological significance. 4.1.2 DNA-binding studies with nuclear extracts. In order to test whether the GRR determined by various genetic analyses (section 3.2) is bound by a protein or proteins, the GCV2 fragment -322 to -248 was amplified by PCR using primers 310F and 260R (section 2.1.4) for use in electrophoretic mobility shift assay (EMSA). This DNA fragment contains the 42 bp region ( -309 to -267) of GCV2 which, when tested for its glycine-responsive function in an heterologous promoter (section 3.3), was shown to include all the important core and flanking sequence for the glycine response. The fragments were labelled during PCR by adding [32P]a-dATP and [32P]a-dCTP instead of unlabelled dATP and dCTP, then isolated after running on a 10% polyacrylamide gel (section 2.6.2). Smaller internal fragments were also generated by cutting with different restriction enzymes. Since the primers used 120 DNA-protein-signalling molecule interactions in the PCR contained Xhoi sites, an internal 42 bp (-309 to -267) fragment could be isolated by cutting then PAGE separation. Two other fragments were generated by cutting the -322 to -248 fragment at an Mboii site (cuts at -295). Nuclear protein extract was prepared for EMSA as described in section 2.8.1 from cells grown in three different conditions: YEPD, Dmin and Dmin containing 10 mM glycine. GTTACCCGGTfGA~CfAGACCCGACATbrcfGACTCtrCTTGAGGTATAJACTCCJCGAGTTCGGAGTCAJGAC -322 t t -248 Xhol (-309) Mboll (-295) X hoi (-267) A B Lanes I 2 3 4 2 3 4 2 3 4 2 3 4 I 2 3 4 5 Figure 4.1. Gel mobility shift analysis of protein binding to the glycine-responsive region of GCV2. Nuclear extract preparation was performed as described in section 2.8.1 and the GCV2 sequences used are indicated in the diagram. A. Protein binding to the -332 to -248; -309 to -267 (internal Xhol fragment); -295 to -248 (Mboll-large fragment); and -322 to -295 (Mboll-small fragment) of the GCV2 gene. The first lane in each set is a no protein control. The second lanes are with the nuclear extract from YEPD-grown cells, the third lanes with Dmin extract, and the last lanes are Dmin with 10 mM glycine extract. 8 fmol of labelled DNA and 5 J.lg of protein were added. B. Proteinase K treatment of nuclear extract. The first and third lanes arc no protein controls, and the second lane is the gel mobility shift assay using untreated nuclear extract of Dmin-grown cells. The fourth and the fifth lanes are with proteinase K-trcated (section 2.2.3) nuclear extract. 8 fmol of labelled Xhol fragment (-309 to -267) and 5 J.lg of protein were used. 121 DNA-protein-signalling molecule interactions The results of EMSA using different DNA fragments are shown in Figure 4.1A. When the nuclear extract was treated with proteinase K before EMSA, complex formation was not observed (Figure 4.1B). This confirmed that the complex formation was due to the interaction of DNA and protein. First, there was one major complex formed with the longer GCV2 fragment ( -322 to -248). A similar complex was obtained using the internal42 bp Xhoi fragment ( -309 to -267). The experiment using the two fragments generated by cutting the -322 to -248 fragment with the Mboii restriction enzyme showed that the longer fragment ( -295 to -248; Mboii-large fragment) formed a complex but the smaller fragment ( -322 to -296; Mboii-small fragment) did not. Although binding occurred to the Mboii-large fragment, it was not as extensive as that to the -322 to -248 fragment or the Xhoi fragment. This indicated that while the protein could bind to the sequence between -295 and -267, for strong binding there was also a requirement for bases in the 14 bp sequence further upstream. These results were consistent with the in vivo results from the deletion and site-directed mutagenesis studies, in which it was shown that while the major control element of GCV2 was located between -289 and -267, 5'-flanking sequences up to -310 were needed for the complete glycine response. It was proposed that the GRR is defined as 5'-CATCN7CTTCTT-3' (section 3.2), which is disturbed by Mboii which cuts within the 5'-CATC-3'. The weaker binding to the Mboii large fragment relative to the Xhoi fragment which contains the intact ORR, suggests that the 5'-CATC-3' is required for stable binding of the ORR-binding protein(s). Secondly, it appears that there are no significant differences in the pattern of band-shifting between the different nuclear extracts from cells grown in YEPD, Dmin, or Dmin containing glycine. Therefore, the ORR-binding protein is constitutively present in cells, and its concentration is not changed by the presence or absence of glycine in the medium. It is therefore expected that a signal is required for this protein to function differentially in the presence or absence of glycine in the medium. Transcriptional regulation of RNA pol II transcribed genes often dependent on the intracellular concentration of signal molecules such as metabolic intermediates. Transcription 122 DNA-protein-signalling molecule interactions factor(s) binding to the GRR of GCV genes may be serving as a signal transducer which regulates the transcription of GCV genes according to the physiological status of the cells. -Xhol fragment Lanes: 2 3 4 5 6 7 8 9 I 0 II 12 13 14 15 16 17 18 19 20 Conditions: c + c + +++++C + Mbo 11-small: 0 0 0 10 10 10 0 s 10 15 20 0 5 10 15 20 (fmol) Figure 4.2. EMSA of Mboll fragments. Two MboJJ fragments (-295 to -248, large fragment; -322 to -295, small fragment) were added alone or together in the binding reaction (from lanes I to 17). Lanes 18 to 20 used the internal Xhol fragment (-309 to -267). Nuclear extract was prepared either from cells grown in Dmin (-)or in Dmin with 10 mM glycine(+) and 5 )lg of protein was used in each lane. The control lanes (no protein) are indicated as "c". 8 fmol of labelled DNA was used for Mboll large fragment and Xhol fragment. The amount of Mboll small fragment added is as indicated in the diagram. Arrow indicates the position of the protein-DNA complex fom1ation. Although no complex formation was observed with the Mboll-small fragment, the possibility cannot be excluded that once the protein binds to sequences in the Mboll large fragment it may further interact with the Mboll-small fragment. Additional DNA sequence between -309 to -295 may be required to stabilise the interaction between DNA and the protein. To determine whether this is the case and to see if there were any differences between the nuclear extracts prepared from cells grown in Dmin and Dmin containing 10 rnM glycine for these interactions, an additional experiment was performed 123 DNA-protein-signalling molecule interactions in which the Mboll-large and small fragments were added in different ratios. If there are any further interactions between protein and the Mboii-large/small fragment, it was expected that there would be changes in the pattern of band shifting depending on the varying Mboll-large and small fragment ratio. Figure 4.2 shows that there is no difference in the shifting patterns with varying Mboii-large and small fragment ratio or with nuclear extracts prepared from cells grown in the presence or absence of glycine. Furthermore, a repeated experiment with each fragment alone did not show any differences. Therefore, it can tentatively be concluded that there is no interaction between the Mboii-large fragment-binding protein and the Mboii-small fragment at least for the protein responsible for this complex formation. No differences were observed with the X hoi fragment using nuclear extracts from Dmin or Dmin with 10 mM glycine as shown in Figure 3.1. From the alignment between the GCV1 and GCV2 genes and site-directed mutagenesis (Figure 3.14) of the GRR, the CTTCTT motif was shown to be the core control sequence. A DNA fragment from -309 to -267 carrying mutations which converted the CTTCTT motif to GGTACC was prepared from pRH16 using primers 310F and 260R (section 2.1.4) which were also used to prepare the wild-type Xhol fragment ( -309 to -267 of GCV2). The labelled Xhol fragment containing the mutated sequences was isolated and it was used for EMSA in comparison with the wild-type Xhol fragment (Figure 4.3A). Complex formation with the mutated DNA was significantly reduced relative to that of the wild-type sequence confirming the importance of the CTTCTT motif. To further test the specificity of the complex formation of the protein with the GRR, competition experiments were carried out using unlabelled wild type sequence or mutated sequence. If the unlabelled sequence competes for binding of the same protein as the labelled DNA, as the concentration of the unlabelled DNA increases, less protein will be available for binding to the labelled DNA fragment. As seen in Figure 4.3 A, it is clear that the unlabelled wild-type sequence competed out the binding of protein to the labelled wild type sequence, whereas mutated sequence did not compete (Figure 4.3 B; this experiment was performed in collaboration with M. Piper, 124 DNA-protein-signalling molecule interactions UNSW). From these results, it is clear that the complex formation is specific to the GRR of GCV2 and the protein specifically binds to the GRR with CTTCTT at its core. It also indicates that some lesser binding can occur through sequences outside the CTTCTT motif. A. Mutant Wild-type sequence B. Fold excess of Fold excess of W.T. competitor DNA mutant competitor DNA 0 1 5 10 50 100 0 5 10 50 100 ~ ...... , ...... free W.T. •••••• DNA •••••• Figure 4.3. Effect of mutation of the CTTCTT sequence (at -286) on protein-DNA complex formation. A. The CTTCTT sequence within the Xhol fragment (-309 to -267) was mutated to GGTACC and tested for binding of protein. The first and fourth lanes arc no protein controls. 20 fmol of labelled DNA and 5 >tg of protein extract were used. B. Competition experiment using unlabelled wild type and mutant sequences. 8 fmol of labelled DNA and 5 >tg of protein extract was used in every lane. Amount of unlabelled competitor DNA was as indicated. 125 DNA-protein-signalling molecule interactions 4.1.3 Effect of in vitro addition of one-carbon metabolites in EMSA In the previous section, it was shown that there was no difference in the binding patterns with nuclear extracts prepared from cells grown under different nutritional conditions. however, it was proposed that there exists a signal for this protein to function differentially in the presence or absence of glycine in the medium. The following work concentrated on the identification of this signal and studying the role of the signal in terms of cell physiology. Most studies of eukaryotic gene regulation are focused on specific regulatory sequences and the proteins that recognise them. However, this approach sometimes overlooks a critical aspect of regulation, which from a biological perspective is the most interesting. Upon changes in physiological state, there must be a signal that initiates the molecular mechanisms that result in altered transcription initiation. The regulatory signal in the present case cannot simply be the appearance of regulatory proteins that interact with the upstream elements. Although transcriptional control is executed by the regulatory proteins that bind to specific DNA sequences, there must be additional regulatory molecules that govern when these regulatory proteins execute their roles in transcription initiation. Therefore, the DNA-binding proteins interact not only with specific DNA sequences but also (directly or indirectly) with signal molecules that distinguish between different physiological states. For example, the expression of galactose inducible genes is mediated by Gal4p which binds to target sites located upstream of GAL structural genes and its negative regulator, Gal80p (Johnston, 1987; Johnston and Hopper, 1982). In this case, the signal molecule is galactose or some direct metabolite of it. Another example is the regulation of the CYCJ gene (Guarante and Mason, 1983; Guarente eta!., 1984). This gene is expressed at high levels when cells are grown in nonfermentable carbon sources, and expressed much more poorly when cells are grown in fermentable carbon sources. It has been shown that intracellular levels of haem may be the signal for its transcriptional control. 126 DNA-protein-signalling molecule interactions The availability of an in vitro binding assay enabled screening for possible interactions between the putative GRR-binding transcription factor(s) and low molecular mass molecules related to one carbon metabolism. We therefore tested a range of compounds that are either products or related intermediates of one-carbon metabolism, including glycine, L-methionine, and tetrahydrofolate (THF). Of the compounds tested, THF had a marked effect on the binding of the protein(s) in the gel mobility shift assays at a final concentration of 1 mM (Figure 4.4). Glycine and L-methionine did not show any significant effect on complex formation even at concentrations beyond normal physiological levels. The range of intracellular concentrations of total free glycine and methionine have been reported to be 3 to 7 mM and 0.4 mM respectively (Cherest eta!., 1973; Messenguy et al., 1980). Compound Glycine Methionine THF Cone. (mM): 0 10 0 lO 0 0 Figure 4.4. Effect of one-carbon metabolites on complex formation in EMSA. Various one-carbon metabolism related compounds were added in vitro in gel-mobility shift assay. Nuclear protein extracts were prepared from cells grown in Dmin. The final concentrations of the reaction were as indicated. 8 fmol of labelled DNA (Xhol fragment; -309 to -267) and 5 !lg of nuclear protein extracts were used. The first lane is a no protein control. The residual uncut DNA ( -332 to -248 of GCV2) from X hoi restriction enzyme digestion can be observed as a higher molecular weight band just above free DNA. 127 DNA-protein-signalling molecule interactions For a detailed analysis, in vitro addition of THF at varying concentrations in EMSA was performed (Figure 4.5 A). It was noticeable that there were two complexes formed with the X hoi fragment in this experiment The higher order complex band may be due to the higher concentration of protein used in this experiment ( 10 Jlg instead of 5 Jlg of protein), which may have caused dimerization. Alternatively, there may be a different protein(s) responsible for this complex formation that is quite unstable such that its activity is lost easily during the nuclear extraction. It can be seen that THF at concentrations between l 0 JlM and 50 JlM led to increased formation of the larger shifted complex (complex II) while the smaller complex (complex I) increased at concentrations of THF between 50 JlM and 0. 1 mM. This increase in complex I appeared to be at the expense of complex II which decreased on addition of THF above 100 JlM (Figure 4.5B). Control experiments adding the same concentrations of buffer and reducing agent (2-mercaptoethanol) needed to stabilise the THF did not affect formation of the DNA-protein complexes (Figure 4.5A). Some of the compounds that are structurally similar and related to THF in one-carbon metabolism were also tested in a similar way. These included folic acid and folinic acid (5-CHO THF). These compounds did not show a significant effect on complex formation even above physiological levels (up to 10 mM). The analysis with folic acid is shown in Figure 4.5C. These results indicate that THF (or one of its derivatives) is the signal molecule which acts as a ligand for the GRR-binding protein. The low molecular mass molecule, THF could affect the affinity of in vitro binding of proteins to the GRR. This provides an insight into the mechanism used by the cell to detect and signal this to the transcriptional apparatus the intracellular availability of glycine which is a donor of one carbon units. Since THF is the basic molecule to which one-carbon derivatives are substituted in their various oxidation states, the control appears to be mediated directly at the level of this compound, or its related derivatives. The putative transcription factor is present constitutively in the cell (Figure 4.1 ), and is activated by modification or ligand binding in the presence of excess glycine in the medium. 128 DNA-protein-signalling molecule interactions A. II I Concentration: 0 SO~M 0.5mM 2mM 0 O.SmM 2mM !O~M O.!mM lmM O.lmM lmM THF 2-mercaptoethanol B. c. 1400 -D- Complex I 2 ·;;; -- Complex II T. = 1000 Cl" -;:; l .S2c. 0 "> -;:; 0::" 4000 Cone.: 0 IO~M lmM 0 ~ ~ ~ ~ ~ ~ ::L E E E E I~M !OO~M !OmM 0 c Figure 4.5. Effect of different concentrations of THF or folic acid on binding of nuclear extract proteins to the Xho! fragment of GCV2. A. The left-hand set of 8 lanes are formation of the complexes using the -309 to -267 (Xhol fragment) of GCV2 at increasing concentrations ofTHF. The first lane is the no protein control. The right-hand set of 5 lanes are controls to which was added different concentration of buffer alone containing 2-mercaptoethanol which is needed for stabilisation of THF. 8 fmol of labelled X hoi fragment ( -309 to -267) and I 0 ~g of protein were used. B. Densitometric analysis of complex I and 11 formation. Relative optical density was measured and error bars represent the standard deviation of at least two separate experiments. C. Effect of in vitro addition of folic acid on complex formation. Different concentrations of folic acid were added to the reaction. The first lane is the no protein control. 129 DNA-protein-signalling molecule interactions In various cell types including S. cerevisiae, the range of concentration of total reduced folates is reported to be 10 to 40 J.!M (Cichowicz and Shane, 1987a; Lor and Cossins, 1972). In the above experiments, the extent of formation of one of the complexes was affected by concentrations as low as 10 to 50 J.!M (complex II), whereas the changes in the other were only detected in the 50 to 100 J.LM range (complex I). While the latter appears to be above in vivo levels, it should be noted that many proteins that bind tetrahydrofolates in vivo have been found to show a greater affinity for the polyglutamylated species, with a binding affinity that decreases as the extent of y glutamyl conjugation decreases (section 1.2.1). It is also not yet clear whether it is tetrahydrofolate, or one of its derivatives that is active in vivo. The use of other metabolic intermediates of THF at different oxidation states is difficult since most of the compounds are extremely unstable and subject to ready oxidation. The use of metabolic mutants which block production of a specific THF derivative would be an alternative way to investigate which derivative of THF is the actual effector. This form of control in which a small effector molecule binds to the DNA binding protein to modulate its activity directly, such as cAMP for E. coli CAP (Ebright, 1993) and tryptophan for the E. coli trp repressor (Somerville, 1992), is less common in eukaryotes, but is seen with some transcription factors. One example of such a mechanism in S. cerevisiae is Leu3p with which a-isopropylmalate acts as a ligand to modulate its transcriptional activity. There are other transcription factors which function in a similar manner. The S. cerevisiae transcription factor, Haplp activates a subset of genes in response to oxygen, such as the cytochrome c isoform-encoding genes, CYCJ and CYC7. Hap I p regulates transcription in response to the cellular haem level by a direct interaction. When haem and Haplp interact, DNA-binding activity is stimulated (Pfeifer et al., 1987a; Pfeifer et al., 1987b). Since oxygen is essential for the production of haem, haem is a suitable effector for transduction of the oxygen status in the cell. The haem responsive domain (HRD) of Hap I p, located C-terminal to the DNA-binding domain, has been implicated in mediating dimerization of transcription factor where upon 130 DNA-protein-signalling molecule interactions it is functional (Zhang et al., 1993). The HRD also appears to mediate interactions between Hap I p and other cellular components since an high molecular weight complex was observed in the absence of haem in EMSA, which prevents Hap 1p from access to its cognate DNA binding site (Fytlovich et al., 1993; Zhang and Guarente, 1994). Under non-limiting haem conditions, Hap I p would be released from the complex and be able to bind and activate its target genes. Saccharomyces cerevisiae responds to pyrimidine starvation by increasing the expression of URA genes, encoding the enzymes of de novo pyrimidine synthesis by a transcriptional activator protein, Pprlp (Lasson and Lacroute, 1980). Purified Pprlp is unable to promote activation of transcription in an in vitro system and transcriptional activation is only observed if either dihydroorotic acid (DHO) or orotic acid (OA) is included in the transcription reactions (Flynn and Reece, 1999). Pprlp directly senses the level of early pyrimidine biosynthetic intermediates within the cell and activates the expression of genes encoding proteins required later in the pathway. In the absence of DHO or OA, the activation domain of Pprlp is constrained in such a way that it is not visible to the transcriptional machinery. Upon binding of either DHO or OA in the activation domain of the protein, Ppr 1p may undergo conformational changes to expose the activation domain, thereby promoting transcription (Flynn and Reece, 1999). Two S. cerevisiae homeodomain proteins, a1 and a2 in the a/a diploid cell form a heterodimer that binds with high specificity and affinity to promoters of many genes for mating, and for regulators of a/a cell specific functions. A recent study has shown that a2 carries a ligand ( a2 tail peptide) that increases the affinity of a 1 for DNA (Stark eta/., 1999), which is a variation of the cases of small molecules that directly activate the binding of proteins to DNA. There are other ligand-binding mediated transcriptional control systems in yeast including the copper-mediated regulation of genes inS. cerevisiae by Ace1p and Maclp. In this case, Cu(I) binding to these transcription factors mediates activation or repression, respectively and for Ace1p, Cu(l) binding leads to folding of the existing protein in to an active form (Winge, 1998). 131 DNA-protein-signalling molecule interactions 4.1.4 foil mutant. To determine whether the effect of THF on the protein-DNA complex formation in vitro is relevant to the control of GCV2 expression in vivo, a mutant strain (YUG 1) which is unable to synthesise THF since it lacks the FOLJ gene, was used. Thisfoll mutant was kindly provided by Dr. 1. H. Hegemann (Dusseldorf, Germany). The FOLJ gene (YNL256W) encodes a protein with three distinct enzyme activities (dihydroneopterin aldolase, 7 ,8-dihydro-6-hydroxymethy lpterin-pyrophosphokinase and 7 ,8-dihydropteroate synthase) which are involved in de novo synthesis ofreduced folate (THF) (J. H. Hegemann, Germany, pers. comm.). A strain lacking this gene (joll) would be severely disturbed in one-carbon metabolism and not viable. The lethality of a foil mutant can be rescued by the addition of 5-CHO-THF (folinic acid) to the media. Folinic acid enters one-carbon metabolism at the level of 10-CHO-THF and the one- carbon unit (formyl-moiety) is subsequently used for purine synthesis and the remaining THF is recycled for use in other parts of one-carbon metabolism (Pasternack et a/., 1994a). 3 Strain- folinic acid 2.5 -o- foil- I 0 )lg/ml 0 foil- 25 )lg/ml 2 -()-- foil- 50 )lg/ml ~ § A foll- I 00 )lg/ml c; 1.5 0 v foll- 250 )lg/ml X FOLJ- 0 )lg/ml + FOLJ- 250 )lg/ml Time (h) Figure 4.6. Growth curve of the foil strain and its isogenic wild-type in Dmin supplemented with various concentrations of folinic acid. Yeast cells were inoculated at an OD600 of 0.05 in glucose minimal medium (Dmin) containing different concentrations of folinic acid. The wild-type strain (FOLJ) was also grown in the absence or presence (250 )lg/ml) of folinic acid. 132 DNA-protein-signalling molecule interactions First, a growth study with the foil mutant was carried out to determine the amount of external THF which is limiting growth of the mutant but still sufficient for cell survival. In Figure 4.6, it was shown that in Dmin containing 250 ~-tg/ml folinic acid this mutant grew at the same rate as the wild-type (doubling time of 1.8 h). Slightly slower growth was observed when the medium was supplemented with 100 ~-tg/ml (doubling time of 2.3 h), and very little growth (doubling time > 24 h) was seen with less than 25 ~-tg/ml folinic acid. When cells were grown in Dmin with 50 ~-tg/ml folinic acid, the mutant grew slowly with a doubling time of 7.4 h, indicating the supply of folinic acid to the cell was limiting, a situation in which intracellular THF concentration will be minimal. Since EMSA of GCV2 with THF indicated that GCV2 gene expression may be regulated by THF, the GCV2-lacZ expression was monitored under this condition (Dmin with 50 ~-tg/ml folinic acid) to elaborate on the role of THF in vivo. To perform the experiment, the foll mutant transformed with the full length GCV2-lacZ fusion (pRHl) was first grown in Dmin plus 50 ~-tg/ml folinic acid to late exponential phase (OD60o of 1.3), then transferred to fresh media; Dmin, Dmin plus glycine (10 mM), Dmin plus folinic acid (100 ~-tg/ml), or Dmin with both glycine and folinic acid, and incubated for a further 2 h, 5 h, or 9 h before harvest for measurement of B-galactosidase activity. In this experiment, two factors were examined for their possible role in the regulation of GCV2 transcription: glycine and THF. Addition of glycine to the medium will change the physiological status of the cells and THF is required to be an effector molecule, which signals the cellular one-carbon metabolic status to exert the regulation of GCV2 gene by acting as a ligand to the putative ORR binding transcription factor(s). GCV2 expression was not significantly increased by addition of 10 mM glycine in media lacking folinic acid (Figure 4. 7) even after 9 h of incubation, indicating that when the intracellular concentration of THF is limiting, the expression of GCV2 did not respond to glycine. This lack of glycine response is not due to malfunction in protein synthesis caused by THF depletion since the cells were able to grow, although slowly, for at least one more generation. Cells assayed after 2 h of incubation in the presence of 133 DNA-protein-signalling molecule interactions folinic acid did not show a glycine response either. In this situation, cells are still limited for THF, therefore GCV2 can not be induced by the presence of glycine in the media. However, after 9 h of incubation, GCV2 gene was induced 2.9-fold. These results support that THF plays an important role in GCV2 regulation in vivo, and in fact, indicate that the GCV2 gene shows a glycine response only when there is sufficient THF present. D Dmin D Dmin + 10 mM gly EJ Dmin + I 00 )lg/ml folinic acid • Dmin +glycine+ folinic acid 0 2 5 9 Time (h) Figure 4.7. Glycine response of the GCV2 gene is mediated in the presence of THF in the cell. The foil mutant (YUG I) transformed with pRHI was first grown in Dmin containing folinic acid (50 )lg/ml folinic acid) up to an OD60Q of 1.3, harvested and washed twice in the sterilised water before transfer to fresh media (Dmin, Dmin plus 10 mM glycine, Dmin plus 100 )lg/ml folinic acid and Dmin plus both glycine (10 mM) and folinic acid (100 )lg/ml). Atier further growing for 2, 5, and 9 h, cells were harvested for B-galactosidase assays. Specific activities represent the mean values of two separate experiments and all errors were less than 20%. In the fall strain, mitochondrial protein synthesis is expected to be abnormal because charged fMet-tRNA (formylmethionyl tRNA) needed for mitochondrial protein synthesis will not be efficiently produced from THF whose synthesis is lacking in this strain. In a wild-type strain, THF is oxidised by the mitochondrial one-carbon metabolic enzymes to produce the charged fMet-tRNA (Figure 1.2). Therefore, the strain was tested for any defect in respiration. It was found that all the fall mutants were unable to grow on nonfermentable carbon sources such as lactate and glycerol. Consequently, an 134 DNA -protein-signalling molecule interactions additional experiment was needed to examine how the GCV2 gene is regulated in a petite mutant (rho- or rho') to see if the result obtained with the fall mutant above is due to the loss of mitochondrial function. Petite mutants (rho') were isolated from Y3 as described in section 2.5.4, and the mutants were transformed with pRHl as single integrants. As shown in Figure 4.8, there was no significant difference between the wild- type cell (grande) and the petite. This result confirmed that the change in the GCV2 expression pattern in Figure 4.6 is solely due to the FOLJ deletion, leading to an inability to synthesise THF. ~ " ~ 750 ..§ 0 E 5 D Dmin 0 500 .B-~ D Dmin + I 0 mM glycine -<: ,_g ·03 250 0. Vl" Wild-lype Petite (Rho+) Figure 4.8. GCV2 -lacZ expression in wild-type strain and petite strain. The yeast strain Y3 and its petite mutant (section 2.5.4) were transformed with pRHl and cells were grown in Dmin to an OD600 of 0.9 - 1.0, then the culture was transferred to fresh Dmin or Dmin with 10 mM glycine. After a further incubation for 2 h, the cells were collected for ~-galactosidase assay. The final OD600 of the culture was 0.4-0.5. It was also investigated whether addition of folic acid to Dmin caused any changes in the expression level of GCV2. Different concentrations of folic acid were added to the medium and GCV2-lacZ expression was monitored. As shown in Figure 4.9, there was no increase of the GCV2 expression by the addition of folic acid to the medium. This result agreed with the fall mutant experiment, in which the addition of folinic acid alone did not cause the elevation of the GCV2 expression (Figure 4. 7). These results probably implied the existence of a mechanism to keep homeostasis of THF species within the cell. 135 DNA-protein-signalling molecule interactions If there is a metabolic cue (i.e. glycine) which shifts this balance, cells may "sense" the change in the level of a certain THF species and restore balance by turning on the appropriate one-carbon metabolic genes such as the GCV genes. 1000 '2 • Glycine ~ 800 ei> D Folic acid E ~ sE 600 !:' .~ 1:l 400 ~ w ;...:: w 200 c. cr." 0 -7 -6 -5 -4 -3 -2 -1 LOG! conccntration (M) 0 Figure 4.9. The effect of folic acid on GCV2 expression. Cells were grown in Dmin to an OD600 of 0.9 - 1.0, then the culture was transferred to fresh Dmin containing different external concentrations of folic acid. The result of effect of the glycine addition is also shown. After a further incubation for 2 h. cells were collected for ~-galactosidase assay. The final OD5oo of the culture was 0.4- 0.5. Errors were less than 20%. 4.1.5 DNA-binding studies with heparin-Sepharose fractions The data shown in Figure 4.5 indicate that there may be multiple protein interactions with the GRR motif and its adjacent sequences. To investigate these potential interactions further, heparin-Sepharose chromatography (section 2.8.1) was used to fractionate the cell extracts to concentrate and partially separate DNA-binding proteins. Yeast strain Y3 (section 2.1.6) was grown in Dmin for this purpose. Figure 4.10 shows the result of EMSA with the GRR of GCV2 (XIwi fragment; -309 to -267) using heparin-Sepharose fractions. The nuclear extract used in Figure 4.5 is also shown for comparison, in this case with a longer exposure to show minor complexes. 136 DNA-protein-signalling molecule interactions 0.1 M 1M IV III II Fraction numbers free DNA nuclear -58 91011121314151617181920 extract Heparin-Sepharose fractions Figure 4. 10. Binding of the GRR to proteins in cell extract fractions separated by heparin-Sepharose chromatography from yeast strain Y3. Proteins were fractionated using an ammonium sulphate gradient as described in section 2.8.1. Gel mobility shift analysis was carried out using 6 ~g of protein from each fraction and I 0 fmol of GCV2 sequence (Xhol fragment; -309 to -267). The first lane (from left) is the no protein control, followed by the nuclear extract from Y3 grown in Dmin, and then by fractions from the hcparin-Sepharose column eluting at increasing salt concentration. One major and three minor complexes are labelled I to IV. The protein elution profile is shown on the right. All of the complexes formed with the nuclear extracts were identified in the heparin-Sepharose fractions, and from Figure 4.1 0, it appears that two of them (complex I and the less abundant II) were due to proteins that were separable. It is clear that most of the complex formation observed using nuclear extracts (Figure 4.1 to 4.4) was of the major complex (I). The other minor complexes (III and IV) were formed in different overlapping fractions. While these may identify further DNA-binding proteins, they may also have been formed in one case (IV) from a multimer of the binding protein from complex I and in the other (III) from a complex containing both proteins seen in complexes I and II. 137 DNA-protein-signalling molecule interactions A. IV III II Free GCV2 Heparin-Sepharose fractions B. IV II Free CCVI Heparin-Sepharose fractions Figure 4. 11. Effect of in vitro addition of THF on heparin-Sepharose fractionated protein. The effect of THF on binding of the heparin-Sepharose fractions to the GCV2 X hoi fragment (-309 to -267; A), and the CCV I sequence from -193 to -162 (B) is shown. For each, the left hand set of ten lanes represents the control without added THF (lane l is no protein control); the right hand sets are the same fractions with l mM THF added to the assay. The availability of heparin-Sepharose fractions enabled an analysis of how the different complexes formed with the GCV2 control region (Xhol fragment) were affected by the presence of the THF. Complex I increased in intensity by up to five-fold on addition of excess THF to most of the fractions, while complex II increased by more than two-fold. The other two complexes were less affected (Figure 4. llA). 138 DNA-protein-signalling molecule interactions Since the GCV1 gene is also regulated by glycine we repeated the above experiments with a 31 bp fragment (-193 to -162) ofGCVJ encompassing the region of strong homology with the GCV2 promoter that contains the core CTTCTT motif. Quantitatively very similar results were obtained (Figure 4. llB) which indicated that the putative GCVJ promoter region binds the same proteins as GCV2 and there is a similar, but less marked, effect of THF on the binding. IV III II Free mutant GCV2 -THF +THF Hcparin-Sepharose fractions Figure 4. 12. Effect of in vitro addition of THF on heparin-Sepharose fractionated protein to the mutated GCV2 fragment. The effect of THF on binding of the heparin-Sepharose fractions to the GCV2 X hoi fragment with mutation of the CTTCTT to GGTACC is shown. The left hand set of ten lanes represents the control without added THF (lane I is the no protein control); the right set is the same fractions with I mM THF added to the assay. When the core CTTCTT sequence was mutated to GGT ACC within the X hoi fragment of the GCV2 promoter, an interesting result was obtained (Figure 4. 12). Consistent with EMSA using nuclear extracts, formation of all four complexes was significantly reduced compared from that seen using the wild-type sequence (Figure 4. 3A). However, upon addition of THF, there was a substantial increase in complex formation, which was the most obvious with complex I, and less marked with complex IV. This result may explain how the construct with mutations in the CTTCTT motif still showed a partial glycine response (Figure 3.14). The addition of THF stabilises the 139 DNA-protein-signalling molecule interactions interaction between the DNA and its binding protein(s), thus overcoming in part the effect of the mutation in the core sequence of the GRR. The same experiment was performed with the GCV2 Mboii-small fragment (-322 to -295), and an interesting result was obtained (Figure 4. 13). Previous results with nuclear extract have shown that no DNA-protein complex formation was found when using the Mboii-small fragment (Figure 4. I and 4. 2). Using heparin-Sepharose fractions containing all four complex formation activities, more detailed analysis could be achieved. fragment - THF +THF Heparin-Sepharose fractions Figure 4. 13 Effect of in vitro addition of THF on heparin-Sepharose fractionated protein to the Mboll-small fragment The effect of THF on binding of the heparin-Sepharose fractions to the GCV2 Mboll-small fragment (-:122 to -295). The left hand set of ten lanes represents the control without added THF (lane 1 is no protein control); the right set are the same fractions with 1 mM THF added to the assay. The relative positions of complexes (I to IV) is shown. As shown in Figure 4. 13, the Mboll-small fragment could form a small amount of complex II which was dramatically increased by in vitro addition of THF. This indicates that there may be an additional protein that binds to the region between -322 to -295 (5' of the GRR) and this protein's binding activity is also affected by THF. No other complexes were formed with the Mboll-small fragment, which is consistent with previous results (Figure 4. I and 4. 2). 140 DNA-protein-signalling molecule interactions 4.2 Footprinting Analysis 4.2.1 Introduction. To verify the GRR determined by in vivo and in vitro studies, footprinting analyses were performed. Footprinting is one approach that can provide detailed information on how a protein binds to DNA. When DNA-protein complexes are treated with DNA cleavage reagents, presumptive sites of bound protein can be deduced by comparing patterns of cleavage to those of DNA alone. DNA cleavage can be achieved by using chemical reagents or enzymes. Two methods of DNA cleavage were employed in this study: hydroxyl radicals formed with 1,10-phenanthroline-copper (OP-Cu); and, DNasei. Footprinting was first devised using enzyme, DNasei (Galas and Schmitz, 1978), which interacts with DNA in both the major and minor groove and has a preference for cutting DNA at a subset of the backbone positions along a DNA strand. Various chemical reagents with the strand nicking activity have also been developed for footprinting analysis such as I, 10-phenanthroline-copper or methidiumpropyl-EDTA iron(II). OP-Cu footprinting has the advantage that it allows in situ footprinting for treatment of the complex in the acrylamide gel (section 2.8.3) which can be very useful, especially when the DNA-binding protein concentration is low. Since only complex I is formed from most of nuclear extracts (section 4.1.2), its formation is most affected by the addition of THF (section 4.1.5), and it was the major one fanned under all conditions it was considered to be the most important in the overall regulation. Therefore, the heparin-Sepharose fraction with the greatest ability to produce complex I formation was used for the footprinting analyses. 4.2.2 DNasel footprinting. Preliminary experiments were required before footprinting in order to quantify the amount of DNA and protein needed. Highly radioactive 32P-labelled DNA (20,000 to 30,000 cpm) is required for efficient footprinting, combined with sufficient protein to minimise background cleavage caused by the presence of unbound DNA. 141 DNA-protein-signalling molecule interactions The 32p end-labelled DNA fragment used in the footprinting assay was prepared first by isolating an EcoRI-Xhoi GCV2 promoter fragment (-351 to -148) from pRHI, then cutting this fragment with AZul, generating an EcoRI-Aiui fragment (-351 to -218). The 5'- EcoRI terminus was labelled using the Klenow fragment of DNA polymerase with [a-32P]dATP and the fragment was isolated by polyacrylamide gel electrophoresis as described in section 2.6.2. Figure 4. 14. Titration experiment. EMSA was performed to determine the amount of DNA and protein to use in footprinting analysis. 2-3 ng of the 32p end-labelled EcoRI Free -··1....: .. :,:. ;·.. Alul fragment ( -351 to -218) of the GCV2 promoter was used DNA • ,. in each lane. Lane 1 is no protein control, lanes 2 and 3 are with 15 1.1 g of protein and lanes 4 and 5 are with 27 1.1 g of Lanes: I 2 3 4 5 protein. A titration experiment was carried out to detennine the amount of protein required for a complete shift of DNA (Figure 4. 14). A complete band shift was observed with the addition of 15 flg of protein. Interestingly, at a protein concentration of 27 f!g, a higher order complex formation was observed. This is thought to be due to dimerization of the DNA-binding protein, and this also indicates that complex III or complex IV observed in Figure 4. I 0 could be the result of dimerization at high concentration of the protein found in complex I. Based on the above result, footprinting analyses were performed (Figure 4. 15) showing that the region from 5'-CATC to CTTCTT-3' was protected. Densitometric scanning also showed that binding of the protein led to an increase in susceptibility to damage of the bases immediately 3' of the footprint (marked as an* in Figure 4. 15), indicating an effect on the topology of the DNA in this region due to binding of the 142 DNA -protein-signalling molecule interactions No Protein Protein r-1 _...... -:::] I 2 3 4 5 - -259 -274 -281 9q () )> 0 q-l q )> () -297 -300 * -305 - -310 - -318 - -323 Figure 4. 15. DNasel footprint of complex I on the GCV2 glycine response r egion. The binding and footprint reacti on was carried out as described in section 2.8.3. A heparin-Sepharose purified protein fraction from strain Y3 was incubated with a 5' end-labelled EcoRl-Aiul GCV2 fragment ( -351 to -218). The first two lanes are free DNA digested with DNasel while lanes 3-5 had increasin g amounts of protein added (15 Jlg, 23 Jlg and 30 Jlg respectively). The sequence protected from DNasel digestion are shown in the box. A densitometry scan of lanes 2 (grey) and 5 (black) is also shown. Numbers indicate the base positions with respect to the start codon. The * indicates the site of enhanced cleavage. 143 DNA-protein-signalling molecule interactions protein. From Figure 3.13B, it can be seen that the protected region corresponds to the sequence of greatest homology between GCVI, GCV2 and GCV3, with the flanking CATC and CTTCTT motifs most conserved. The protein binding to this region is therefore an excellent candidate for a transcription factor that mediates the glycine control. ,I Lanes THF cone . 7 • 6 lmM 5 0.5mM 4 50~M 3 !O~M 2 O~M G+A Figure 4. 16. Effect of THF in DNasel footprinting of the complex I. An heparin Sepharose purified protein fraction from strain Y3 was incubated with a 5'-labelled EcoRI-Alul GCV2 fragment ( -351 to -218) in the absence or presence of THF. The first lane is the G+A sequencing ladder, lanes I and 7 are no protein controls (free DNA) with different amount of nuclease added (2 units for lane 1 and l unit for lane 7) and lanes 2-6 had increasing amounts of THF added as indicated. Each reaction contained 23 ~g of protein. A densitometry scan of lanes 2 (grey) and 5 (black) is also shown. The footprint reaction was carried out as described in section 2.8.3. 144 DNA-protein-signalling molecule interactions These results are fully in accord with the genetic data presented in Chapter 3 since the binding of the protein depends on the region containing the CTTCTT motif, but is augmented by bases further upstream to include the CATC. It can be seen that the region around the CTTCTT motif is protected preferably at a lower concentration of protein ( 15 Jlg), but the protection region is further extended to include 5'-CATC-3'. This becomes clearer at higher concentration and may be the result of protein dimerization as suggested for the band-shifting pattern in Figure 4. 14. To investigate the effect of THF on the binding of the protein to the DNA, THF was added at different concentrations during the DNA-protein binding reactions before being treated with DNasei. Although precise data analysis was hampered by the difficulty of even loading of the samples and of the cutting in the absence of added protein, an interesting point was raised by the result. The DNasei protected region of the 500 JlM THF-added sample was compared with that of the no THF-control by densitometry (Figure 4. 16). There was further protection in the region 5' of the GRR (up to -308) when THF was added. It is not clear whether this is due to the effect of THF on complex I formation or due to another effect such as formation of complex II, because it is possible that a residual protein activity responsible for complex II formation may be affected by THF addition to effect increased binding. Further footprinting analysis with heparin-Sepharose fractions containing complex II activity is required to test this possibility. 4.2.3 OP-Cu (1,10-phenanthroline-copper) footprinting. Since DNasei is roughly the same size as the DNA-binding protein it is detecting, the DNA region covered by the protein is often overestimated. In addition, DNasei has a preference for cutting DNA at only a subset of the backbone positions along DNA. Use of less bulky chemical reagents has allowed higher resolution of protection by protein DNA complexes (Dixon et al., 1991). One such reagent, 1,10-phenanthroline-copper (OP-Cu) complex was employed for footprinting analysis of the GRR of GCV2. The chemistry of the reaction can be summarised as follows; 145 DNA-protein-signalling molecule interactions (OP)2Cu2+ + R-SH (3-mercaptopropionic acid) <---> (OP)2Cu+ + 112 (RS-SR) (1) 2(0P)2Cu+ + 02 + 2H+ ---> 2(0P)2Cu2+ + H202 (2) (OP)2Cu+--DNA + H20 2 ---> (OP)2Cu2+-0H--DNA + OH- (3) The DNA strand cleavage activity of OP-Cu depends on the production of hydrogen peroxide. Reduction of the cupric ion from the thiol group of 3- mercaptopropionic acid results in the OP-cuprous complex (reaction 1). This is then oxidised by molecular oxygen generating hydrogen peroxide and reforming the cupric complex (reaction 2). When the OP-cuprous complex is bound to the surface of DNA, a one-electron oxidation of the cuprous complex by hydrogen peroxide occurs and this attacks the C-1 or C-4 hydrogen of the deoxyribose moiety to cleave DNA (reaction 3) (Sigman eta/., 1991 ). To avoid overdigestion, the nuclease activity must be efficiently quenched. 2,9-dimethyl-1, 10-phenanthroline blocks the nuclease activity by sequestering all the available copper in an ine1t form. Figure 4. 17. OP-Cu footprinting of complex I. An heparin-Sepharose purified protein fraction from strain Y3 was incubated with 2-3 ng of 32P-labelled EcoRJ-Alui GCV2 fragment (-351 to -218) in the absence or presence of THF. The first lane is the G+A sequencing ladder and lanes 5 and 6 are no protein controls (free DKA). Lanes I and 2 were without added THF and lanes 3 and 4 were with THF (l mM) in the binding reaction. 23 ~g of protein was used in lanes l to 4. The footprint reaction was carried out as described in section 2.8.3. G+A I 2 3 4 5 6 146 DNA-protein-signalling molecule interactions Figure 4. 17 shows the result of OP-Cu footprinting of complex I. The result in the absence of tetrahydrofolate (THF) was very consistent with that of DNasei footprinting in Figure 4. 15. When no THF was added in the DNA-protein binding reaction prior to DNA cleavage (lanes I and 2), the 5' limit of the protected region was the 5'-CATC-3' and the 3' end of the region included the CTTCTT motif. These results confirmed the GRR binding site. Interestingly, when THF was added in the DNA-protein binding reaction, no protection occurred (lanes 3 and 4; Figure 4. 17). This is probably due to the fact that THF or its derivative that acts as a ligand for the GRR-binding protein are easily oxidised during the chemical cleavage reaction. This supports that THF is important in the binding of protein to the GRR. It may also indicate that products from the oxidation of THF may actually lead to inhibition of binding of the protein to the GRR since a footprint was obtained in the absence of added THF. In this chapter, analyses of interactions between the GRR and the proteins binding to it were performed. Results of EMSA with DNA fragments spanning different regions of the GRR were very consistent with the deletion analysis in chapter 3 and footprinting further confirmed that the GRR is 5' -CA TCN 7CTTCTT-3'. Multiple complexes were observed to form between DNA binding proteins and the GRR, and in vitro addition of THF was shown to increase the extent of this binding, which indicated that THF (or one of its derivatives) is probably acting as a ligand for the GRR-binding transcription factors to influence the GCV2 gene expression. This was supported by the GCV2 gene expression study using a mutant that is unable to synthesise THF. Therefore there appears to be a transcriptional system that specifically regulates one-carbon metabolism controlled by its intermediate (THF or its derivative). This system is therefore much wider in its effects than just the genes involved in glycine metabolism, and the question arises of how extensive this system is. In the next chapter, attempts were made to determine the extent of this regulatory system using genome-wide analysis of transcriptional control. 147 Genome-wide transcript analysis Chapter 5: GENOME-WIDE ANALYSIS OF ONE-CARBON METABOLISM 5.1 Introduction Biology has entered the era of genomics: genome sequencing projects have generated and will continue to generate enormous amounts of sequence data. The genomes of several bacteria including archaebacteria and that of S. cerevisiae have already been completely sequenced (Goffeau, 1997), and the sequence of the yeastS. cerevisiae genome has provided the first complete inventory of the working parts of an eukaryotic cell. The challenge is now to elucidate what each gene product does and how they interact to respond to environmental changes, develop, grow, and divide by investigating gene expression patterns on a whole genome scale. A wide range of experimental approaches have been developed to allow functional genomics to establish an integrative view of the working parts inS. cerevisiae. In order to determine the function of novel yeast genes, the European Functional Analysis Network (EUROFAN) has been established (Oliver, 1996). The first aim was to produce a complete set of single-gene deletion mutants covering all the open reading frames (ORFs) revealed by the completeS. cerevisiae genome sequence. This mutant collection will represent a major resource, not only for the functional analysis of the yeast genome but also for facilitating the analysis of genomes from higher eukaryotes by permitting their functional mapping onto that of yeast through transcomplementation experiments (Oliver et al., 1998). Another route to the elucidation of gene function is the analysis of all yeast proteins synthesised under a given set of conditions, the so-called proteome (the PROTEin complement expressed by a genOME) by using two-dimensional gel electrophoresis. Identification of proteins can be achieved by various methods, including peptide mass finger printing and chromatographical determination (Wilkins eta!., 1996). 148 Genome-wide transcript analysis Another powerful tool in the elucidation of gene function is the quantitative analysis of gene expression levels on a genome-wide scale under a variety of physiological or developmental conditions. This is to determine the complete set of yeast genes expressed under a given set of conditions (the transcriptome). Knowing when and where a gene is expressed often provides a strong clue to its biological function, and conversely, the pattern of genes expressed in a cell can provide detailed information about its state. Although regulation of protein abundance in a cell is by no means accomplished solely by regulation of mRNA, virtually all differences in cell type or state are correlated with changes in the mRNA levels of many genes. Establishment of the global and quantitative mRNA expression profiles allows a quantitative description of the biological system. One approach to such an analysis of gene expression is the use of SAGE (serial analysis of gene expression) technology to measure the number of copies of a given mRNA per cell using a short sequence tag (9-11 bp) that contains sufficient information to identify a unique transcript (Velculescu et al., 1997). However, work-up of this method is complex and statistical limitations apply when analysing the results. A better approach is the use of hybridisation-array technology. This method uses DNA-arrays on glass or nylon support containing almost all of the yeast ORFs, synthesised by PCR using primers complementary to the ends of ORFs, which are then hybridised with labelled eDNA probes prepared from total mRNA of cells (Nguyen et al., 1995; Schena et al., 1995). In spite of many advantages for large-scale measurement of gene expression, the use of this method has been restricted by the relative inaccessibility of the instrumentation needed for the preparation of DNA-arrays. Recently, rniniarray membranes (GeneFilters 1M) have become commercially available (Research Genetics, Inc., U.S.A.), and these have made the use of the powerful hybridisation-mTay technology more accessible. These rniniarray membranes were employed in this study to investigate the global expression profiles of yeast cells under two different growth conditions (minimal medium with or without glycine) to gain an insight into the genome-wide transcriptional control of genes mediated by the glycine response. This has 149 Genome-wide transcript analysis two main aims: (i) to identify all of the genes subject to control by glycine, and (ii) to identify, if possible, genes encoding functions in one-carbon metabolism that have yet to be fully characterised (e.g. glycine/serine transporter(s) from the mitochondrion, and transcription factors involved in the glycine response). 5.2 Genome-Wide Transcriptional Analysis 5.2.1 Methodology One approach to understanding physiological mechanisms is to compare patterns of gene expression associated with varying physiological states (in this study, Dmin and Dmin with 10 rnM glycine). Comparisons of gene expression patterns can provide insights into the action and interaction of genes that are important in the response to specific environmental stimuli or during specialised processes. Furthermore, comparison of transcriptomes from a variety of physiological states should provide a minimum set of genes whose expression is required for normal growth. To compare the gene expression patterns of cells grown in Dmin and Dmin plus glycine (10 rnM), miniarray membranes were used in this study. Miniarray membranes (GeneFilters™, Research Genetics, U.S.A) consist of two filters containing a total of 6144 yeast open reading frames (ORFs) which were individually amplified by PCR. Each PCR product contains the complete ORF including the start and stop codons. The DNA was then denatured, spotted on a positively charged nylon membrane and UV cross-linked to the membrane. Positive controls consisting of total yeast genomic DNA were also printed on each membrane. Labelled eDNA probes used in the hybridisation were prepared from mRNA of yeast strain Y3 as described in section 2.7.3. The mRNA samples were from yeast grown in two different conditions, Dmin and Dmin containing 10 mM glycine up to an OD60o of 0.5. Gene-array analysis was performed as described in section 2.7.6. After the filters were 150 Genome-wide transcript analysis • ·'!, .. ~... ~. .!!L Dmin Dmin+gly Figure 5.1. Miniarray membrane hybridisation. Comparison of the hybridisation patterns with labelled eDNA prepared from mRNA of cells grown at log phase (OD6Qo of 0.5) in two different conditions; Dmin and Dmin with 10 ruM glycine. Two membranes containing 6144 yeast open reading frames were used in the hybridisation. The data shown are from a single filter which was first probed with eDNA prepared from cells grown in Dmin, then stripped and reprobed with eDNA from cells grown in Dmin with glycine. Every ninth row (indicated by arrows) is a hybridisation control lane containing yeast total genomic DNA. Each dot on the membrane represents a single gene's expression. exposed to a phosphor screen and analysed using a Phosphoimager (Bio-Rad), raw images of the array-hybridisation were acquired (Figure 5.1). For the convenience of analysis, the hybridisation images of the two different conditions (Dmin and Dmin with 10 mM glycine) were superimposed. A colour was arbitrarily allocated according to nutritional condition; Dmin was red, and Dmin with 10 mM glycine was allocated blue (Figure 5.2). Therefore, after superimposition, red spots indicated that the level of a gene's expression in cells grown in Dmin with I 0 mM glycine was higher than that in Dmin and vice versa for blue spots. If the level of a gene's expression was similar in both conditions, the additive colours showed as a yellow spot. !51 Figure 5.2. Superimposed colour representation of the hybridisation membranes. Signals of genes expressed more in Dmin than in Dmin with 10 mM glycine are represented as red dots while blue dots indicate genes expressed more in Dmin with glycine than in Dmin. Yellow dots indicate that the gene was expressed at a similar level in both conditions. Genome-wide transcript analysis 5.2.2 Functional sorting of the transcriptome. Functional genomics requires analytical strategies that are comprehensive and hierarchical. Comprehensive because it is the aim to uncover the action and interaction of all genes and hierarchical because this overwhelming task is only possible by grouping genes of related function (Oliver et al., 1998). Having achieved such a grouping, it is then possible to construct subgroups to achieve a closer approximation to the function of each novel gene. This allows identification of sets of genes influenced by the physiological event or a particular mutation and could ultimately allow an understanding of the transcriptional program of the cell. The global view of changes in expression of genes encoding pivotal enzymes can provide insight into metabolic reprogramming, and the behaviour of large groups of functionally related genes can provide a broad view of the systematic way in which the yeast cell adapts to its changing environment. By comparing the mRNA expression profiles of cells grown in Dmin and Dmin supplemented with I 0 mM glycine, 191 genes were shown to be either increased or decreased in their expression levels by more than two-fold. Experiments were performed using the same set of mini array filter membranes to avoid potential variation from one filter to another due to different DNA loading onto the filter. A quantitative analysis using a single filter under the same condition showed that hybridisation signals are highly reproducible even after several reusing times; a linear correlation coefficient between two separate hybridisation was 0.964 and the statistical probability of the two data sets being different was less than 0.0001 (Cox et al., 1999). The experiments here were repeated using the same filters and the results were qualitatively similar. Since signals for some genes were interrupted by strong signals of neighbouring genes on the membrane, a visual search using the two colour images also aided identification of genes with altered expression. The diffusion of the signals seen in Figures 5.1 and 5.2 can be avoided if the probe labelling is performed with a 33P-labelled nucleotide instead of 32P-labelled nucleotide since 33p produces higher image resolution due to its lower energy !52 Genome-wide transcript analysis beta emission. At the time of this experiment, only the 32P-labelled nucleotide was available and hence some spreading of strong signals occurred. Nonetheless, among those 191 genes identified, 159 genes increased in expression and 32 genes showed decreased expression following glycine supplementation of minimal medium. Grouping of these genes by related function was carried out and the result were as follows (also see appendix): • 39 genes (20%) were of unknown function; • 22 genes were for proteins related to various metabolic pathways including one-carbon and amino acid metabolism; • 24 were related to transcription; • 17 were related to cell wall or membrane synthesis/structure; • 11 were related to transporters (including major facilitator proteins); • 15 were for stress-related proteins; • 10 were related to signal transduction; • 17 were related to protein synthesis or breakdown. Regarding the range of regulation observed in this analysis, the maximum extent of induction from Dmin to the Dmin plus glycine condition was approximately 140-fold (PAU5; encoding protein with similarity to members of the PAU I family also called seripauperins; Viswanathan et al., 1994) and the maximum reduction was approximately 90-fold (YOR364W; encoding protein of unknown function). The above results need more detailed experiments to quantitate accurately the changes in expression level seen and to confirm the genes that were being expressed differently. This work, using a time course analysis and 33P-labelled probes is currently under way in collaboration with S. Winata (Honours student, UNSW). 5.2.3 One-carbon metabolism Among 22 genes that were classified as encoding proteins related to metabolic pathways, 13 could be subgrouped as genes encoding proteins for one-carbon metabolism or with closely related functions. All the genes identified in this group showed higher !53 Genome-wide transcript analysis expression levels in a glycine-supplemented condition. Changes in the expression of genes with known functions provide an insight into the way the cell adapts to its changing environment. As expected, the expression of all the CCV gene was increased in Dmin with I 0 mM glycine. For comparison, the inductions observed in this analysis for CCV1 and GCV3 were 2.7-fold and 2.2-fold respectively, whereas from the lacZ fusion study (Figure 3.5) these were 8.2-fold and 3.2-fold induction respectively. It is not clear why the DNA arrays gave relatively lower induction levels, although there were differences in time of exposure of the cells to glycine (18 h for gene-array analysis versus 2 h for lacZ fusion study). We have noted similar underestimation for the extent of induction of other genes from micro-array data form other sources (Schena et al., 1995), including that for the LPDJ gene following shift of cells from fermentative to respiratory growth. Despite these quantitative differences, this result confirmed that the array-hybridisation analysis using this miniarray membrane worked qualitatively and could detect changes seen in the previous detailed analysis of the CCVI, GCV2 and GCV3 genes. One-carbon metabolic flow. From the genes that had altered expression levels, it was possible to glean some insight of the flow of metabolites in the cell. Figure 5.3 shows the one-carbon metabolic flow in yeast when cells were grown in glycine-supplemented medium. Once glycine enters into the cell, it can be freely transported into the mitochondrion (Cybulsky and Fisher, 1976). From growth and NMR studies with various one-carbon metabolic mutants, it was suggested that the GDC and mitochondrial SHMT channel glycine through sequential reactions (Figure 3.1) to yield serine which can be utilised in the cytoplasm (McNeil eta/., 1996). Serine in the cytoplasm then can be used for the synthesis of 5,JO-CH2-THF, a key one-carbon metabolic intermediate, via the reaction of cytoplasmic SHMT which is encoded by SHM2. A study with S. cerevisiae has shown that SHMT formation was stimulated six-fold when cells were grown with added glycine at 10 mM concentration (Botsford and Parks, 1969). 5, IO-CH2-THF is an important branch point in one-carbon metabolism since it can be used !54 homocysteine r------> F ~------,~ serine r glycmc cleavage S)-Stcm THF "'"" ~ ... ~ glycine t~ serine 4:.,. DHF ~ \ lBI'II ~ . 64 I I DIIIJ ~ g l yc ine ~_ ... 5 , 1 0-CH~THF ( ) Thym;dylate ) ...._IBIII=--...~-'7-~,~"" 5,10-CH2·THF 1- -~gly·"'" ci ne phophatidyl ;;3~3+4 spermine ".. -choline t NADH 5, I 0-CH-THF 5 , 10-CH-THF1 ~ MITOCHONRION "H(JIM! 5-CHO-THF 1~ . 97 t I• 10-CHO-THF t IO- rO- t ..£ formate \.. ~ ---- -.;J ~ - ' ----.....,...--....------./ ------). formate Figure 5.3. One-carbon metabolic flow in yeast when cells were grown in the presence of extracellula r 10 mM glycine. Genes that showed increased expression in Dmin wi th 10 mM glycine are boxed and numbers indicate fo ld increase. Only the relevant pathways are shown in the figure. CCVI, GCV2 , and GCV3, encode the subunits of the GDC; SHM2, cytoplasmic serine hydroxymethyltransferase; DFRJ, dihydrofolate reductase; MTDJ, monofunctional NAD+-dependent CH2-THF dehydrogenase; ADEJ 3, adenosylsuccinate lyase; APTI , adenine phosphoribosyltransferase; SAM I , S-Ado-methionine synthetase; OP/3, phospholipid N methyltransferase; SPE4, spermine synthase. Thicker lines indjcate the now of one-carbon metabolites. Genome-wide transcript analysis either for methionine synthesis via S,lO-CH2-THF reductase, thymidylate synthesis via thymidylate synthase, or for de novo purine synthesis via more oxidised one-carbon molecules (Figure 1.2). From the DNA microarray analysis, it appears that the pathways for purine and methionine synthesis and utilisation were stimulated by excess glycine in the medium. Three genes for each pathway have been identified (Figure 5.3). The increase of the MTD 1 transcript in Dmin with glycine medium was interesting, since this gene encode an N AD+ dependent CH2-THF dehydrogenase that is involved in the flow of one-carbon units in the oxidative direction. In contrast, C1-THF synthase is involved in both the oxidative and reductive directions (West et al., 1996). This indicates that in the presence of excess glycine, one-carbon metabolic flow shifts toward the more oxidised intermediates and promotes de novo purine synthesis. Identification of YER183C. Another very interesting finding was that the transcript of YER183C showed a major increase (about 13-fold) in cells grown in Dmin with glycine. The function of the protein encoded by this gene has not been studied inS. cerevisiae, however, its deduced amino acid sequence shows greatest similarity to human 5,10-CH-THF synthetase (also called 5-CHO THF cycloligase), while also showing homology to 5,10-CH-THF synthetases from other organisms such as rabbit and C. elegans. There is 30% identity and 52% similarity between the human and yeast proteins (Figure 5.4). Only recently has a metabolic role been assigned to 5-CHO-THF despite the fact that it has been found to comprise about 10 to 25% of the intracellular folate pools of most organisms (Cossins, 1984). Because of its stability (to oxygen and alkaline pH), it has been suggested that this derivative may function as a storage form of folate in cells that have a dormant stage, such as spores or seeds, or may even play a regulatory role in one-carbon metabolism (Kruschwitz et al., 1994). 5,10-CH-THF synthetase is the only enzyme to use 155 Genome-wide transcript analysis 5-CHO-THF as a substrate and irreversibly catalyses the reaction (Hopkins and Schirch, 1984): 5-CHO-THF + ATP ---> 5,10-CH-THF + ADP + Pi The 5,10-CH-THF synthetase activity has been shown to be present in a wide variety of cells (Horne et al., 1989). 10 20 30 40 50 60 S.cerevisiae MAT------KQLLRRQIKRVINALDYDIIAAESHTISQAVRSLIASANSRRVACYMSMDK human MAAAAVSSAKRSLRGELKQRLRAMSAEERLRQSRVLSQKVIAHSEYQKSKRISIFLSMQD * ** * * .*. ,** * * * ** S.cerevisiae GEVTTGEIIKNLFQDGQEVFLPRCTHTSESKHFKLREDHHPHLIFHRMSSLKMVFDLKPQ human -EIETEEIIKDIFQRGKICFIPR--YRFQSNHMDMVRIESP----EEISLLPKTSWNIPQ * * **** ** * * ** * * * * * ** S.cerevisiae GPYQLKEPEPHIEESDILDVVLVPGVAFDIKTGARMGHGAGYYDDFFQRYKILHEGQKPL human -PGEGDVREEALSTG-GLDLIFMPGLGFD-KHGNRLGRGKGYYDAYLKR-CLQHQEVKPY * * ** ** ** * * * * * **** * * ** S.cerevisiae LVGLCLMEQVASPIPLEKHDYSMDCIVCGDGSIHWFQ human TLALAFKEQICLQVPVNENDMKVDEVLYEDSSTA--- • •• . .. • • • • Figure 5.4. Amino acid sequence alignment of human 5,10-CH-THF synthetase and the deduced sequence from S. cerevisiae YER183C gene. Asterisks indicate identities and dots indicate similarities between two sequence. Lines represent gaps that were introduced to provide the best fit. A second catalytic activity of SHMT has been described as the irreversible hydrolysis of 5,10-CH-THF to 5-CHO-THF (only occurs in the presence of glycine, forming a SHMT- glycine complex). E. coli lacking SHMT were shown not to contain intracellular 5-CHO THF, demonstrating that this reaction is probably the physiological source of 5-CHO-THF in vivo (Stover and Schirch, 1992; Stover and Schirch, 1990). It was also shown that 5-CHO THF is an inhibitor of folate-dependent enzymes including the SHMT, suggesting it plays a role in the regulation of one-carbon metabolism affecting glycine, purine, thymidy late synthesis, and homocysteine remethylation (Girgis et al., 1997; Stover and Schirch, 1993). 156 Genome-wide transcript analysis In humans, glycine is an effective one-carbon source for the synthesis of 5-CH3-THF and subsequent homocysteine remethylation (Girgis et al., 1997). 5-CHO-THF was suggested to be required to keep cytoplasmic folate metabolism in homeostasis by mediating the flow of one-carbon units in the cytoplasm through either the homocysteine remethylation or purine synthesis pathways. 5-CHO-THF regulates cytoplasmic SHMT by inhibition of folate-dependent syntheses. This may be a mechanism used by the cell to prevent accumulation of intracellular folate as 5-CH3-THF and thereby redirect one-carbon units for other metabolic pathways such as purine biosynthesis (Girgis et al., 1997). Similar regulatory mechanisms may also apply in S. cerevisiae. With an excess of intracellular glycine, 5-CHO-THF would be produced by the second catalytic activity of SHMT due to higher SHMT activity (increased SHMT mRNA level in medium containing 10 mM glycine). The 5-CHO-THF may enter the purine biosynthetic pathway via the product of YER183C, generating 5,10-CH-THF which is further oxidised to 10-CHO-THF in a reaction promoted by increased activity of NAD-dependent CH2-THF dehydrogenase encoded by MTD 1. In this way, the protein encoded by YER 183C may play an important role in balancing the flow of the one-carbon units in the cell. In support of this hypothesis, a 13C NMR study showed that the formyl group of 5-CHO-THF was first converted to 10- CHO-THF, most of which was subsequently used in purine synthesis (Pasternack et al., 1996). Although the functionality of the putative protein encoded by YER183C should be tested prior to examining the above hypothesis, the identification of YER183C from the gene array analysis provided an insight into the control of cellular metabolism in response to the changing environment. It also illustrates the power of this hybridisation-array technology not only for building on the knowledge of the regulation of already known genes in different conditions, but also for identifying candidates for further studies. 157 Genome-wide transcript analysis 5.2.4 Global metabolic flow The phenotype of an organism is largely determined by the genes expressed within it. This information builds a transcriptome conveying the identity and level of each expressed gene for a population of cells. Unlike the genome, which is essentially a static entity, the transcriptome can be modulated by both external and internal factors. The transcriptome thereby serves as a dynamic link between an organism's genome and its physical characteristics. In addition to the one-carbon metabolism related genes, there were a large number of other genes that showed increased transcription levels in glycine-supplemented medium. An attempt has been made to explain the changes in expression level of genes that are related to other metabolic pathways to provide new information useful for understanding numerous aspects of cell biology and biochemistry. Serine produced from glycine by the cooperative reaction catalysed by the GDC and mitochondrial SHMT can be utilised for cytoplasmic one-carbon metabolism as described in the previous section. An alternative pathway for the utilisation of L-serine is via the reaction catalysed by L-serine dehydratase (SDH), resulting in the formation of pyruvate (Snell, 1984). In fact, a recent in vitro and in vivo study ofL-serine metabolism in rat liver showed that SDH is the major path for L-serine metabolism, and the pyruvate produced is used either for gluconeogenesis or for the TCA cycle (Xue eta!., 1999). The gene-array analysis showed that among those genes which had expression levels altered by more than two-fold, some are related to amino acid biosynthesis (see Appendix). Combined with L-serine metabolism via SDH to produce pyruvate, a more global metabolic flow within the cells grown in the presence of excess glycine, can be suggested as depicted in Figure 5.5. Serine-derived pyruvate may be converted to citrate via the pyruvate dehydrogenase complex and citrate synthase. The genes for both of these enzymes showed increased expression in the glycine-supplemented medium. Isocitrate, the isomerised form of citrate, 158 lysine pyrimidine aspru;agine , 1 m!8!el§lldm 2. 19 i ""' f::; NH4+ h omocysteine-e~.----- h · ~ aspartate methionine PYJ''· 1'.' nl\ all' threonine 11<-h.' d ro~l ' lla'l' ~ acetyl-CoA isoleucine / oxoloaccJAIP. .... t .... SAM ...... I ' .... arginine .... malate .... citrate • 'IQII ...... ,:. ' ~ ;,serine phophatidyl spermine ~oxylate ", .- THF ~ DHF) ( ..,_~~~~~~~~~~~=-- isoci trate THF . k. 3 54 fumarate 5.94J glycine ~ senne · lllll!lllliil' :~~-TI!Fo( )o Thym;dyla ' 2 \ a-ketoglutarate __ ~ glycine ~NAD+ \ 1 succinate / • ~ ~ glycin-* IIIJD3.34 ~ CH -THF ~succi n y l__./' 2 NADH Co-A CH-THF ~ ijlflt>jii 5-CHO-THF CH-THFt MITOCHONDRION t 10-CHO-THF l~CtT~------IJ!JIIIJIIINf---~)o pu~ t -t- 293 ·~ format~ ------~ formate Figure 5.5. Global metabolic flow when yeast cells were grown in medium containing 10 mM glycine. Genes (or proteins encoded by the cognate genes) which showed increased expression in Dmin with 10 mM glyc ine are boxed (accompanying numbers indicate fold increase). Only relevant pathways are shown in this fi gure. Names of proteins encoded by one-carbon metabolism related genes are shown in Figure 5.4. Genome-wide transcript analysis has two major fates. When cells need energy, it is oxidatively decarboxylated to 2- oxoglutarate, and when energy is abundant, it enters the glyoxylate cycle via isocitrate lyase for the formation of biosynthetic intermediates such as oxaloacetate from which many amino acids are derived (Figure 5.5). In the presence of glycine, the glyoxylate cycle appeared to be more active because the mRNA transcript level of ICLJ (encoding isocitrate lyase) increased about six-fold. In this fashion, serine which was derived from glycine via one carbon metabolism may contribute to amino acid biosynthesis. The amino acid biosynthetic pathways may also be eventually connected to one-carbon metabolism via the methionine biosynthetic pathway (Figure 5.5). The amino acids synthesized could be subsequently used for the production of other cellular proteins which may be needed for adapting to a changing environment; 14 genes identified from this gene-array analysis were related to protein synthesis (see Appendix). From these results, it is hypothesised that the addition of glycine activates not only the one-carbon metabolic pathway, but also other metabolic pathways such as the glyoxylate cycle and various amino acid biosynthetic pathways. This provides a glimpse into the mechanism whereby cells balance cellular metabolism in response to an altered environment caused by the addition of a single compound (glycine). 5.2.5 Transcription-related genes Previous sections have concentrated on metabolic flow within the cell when glycine was added to the medium. The gene array analysis also identified other groups of genes whose transcript levels were altered in response to glycine addition. One of the functional groups was transcription related. It is conceivable that the proteins encoded by this group of genes are required to adjust the expression level of other genes, necessary for adaption to the surroundings. This also may be helpful for the identification of the potential transcription factors which are responsible for, or related to the glycine response of the GCV genes and/or other one-carbon related genes. In this section, selected genes with known functions were discussed to explain their potential role in response to glycine in the medium. !59 Genome-wide transcript analysis Among those with an altered expression with glycine in the medium, the GALli gene was shown to increase expression level. Galli p is a component of a multi-subunit mediator complex of RNA pol II holoenzyme inS. cerevisiae (section 1.5.1). The mediator complex is known to be a coactivator that enables the basal transcription machinery to respond to gene-specific transcription regulatory proteins to cause activation or repression. Different sets of mediator proteins may function in distinct transcriptional pathways (Han et al., 1999). The Galllp-mediator protein may interact with the transcription factor(s) that is/are responsible for the glycine response of the one-carbon related genes, including CCV genes, to control their expression by modulating RNA pol II activity. Transcription of the M CM 1 gene was also increased by the addition of glycine. Mcmlp is an essential multifunctional transcription factor involved in a variety of cellular processes including the transcriptional regulation of cell cycle control (Althoefer et al., 1995), cell-type determination, the pheromone response (Dolan and Fields, 1991; Treisman and Ammerer, 1992), cell wall and membrane integrity, cellular metabolism (Kuo and Gray hack, 1994; Messenguy and Dubois, 1993), and stress tolerances (Kuo et al., 1997). Mcmlp recruits specific cofactors to the promoters at which it acts and the type of regulation imposed by Mcm I p depends on the cofactor recruited. The identification of this gene is interesting in that many genes identified in the gene-array analysis are related to cell wall I membrane integrity and stress response, as well as to cellular metabolism. Mcmlp may be needed directly or indirectly for transcriptional control of these cellular processes for adaption to the changing environment. F-box proteins are involved in the ubiquitin-dependent proteolytic pathway targeting many key regulatory proteins for rapid intracellular degradation. Many F-box containing proteins are directly or indirectly implicated in transcriptional control. Additionally, many have been identified by the genome sequencing project and it is thought that these proteins probably participate in the regulation of cellular processes such as cell division, signal transduction and development (Patton et al., 1998). Four genes encoding F-box containing 160 Genome-wide transcript analysis proteins have been identified from this analysis, two of them with unknown function. It is not certain whether these proteins are directly or indirectly involved in the regulation of cellular processes in response to glycine, however, it is possible that they are required to integrate many metabolic pathways and thus identification of this group of genes may provide potential candidates for further studies. In addition, this analysis revealed three genes that encode proteins with unknown function containing either a zinc-finger or leucine-zipper motif. These motifs are commonly (although not exclusively) found in many transcription factors. Therefore, these are good candidates for further study to investigate whether they are involved in regulating genes for the glycine response. 5.2.6 Other functional groups This gene-array analysis also identified other groups of genes that were categorised as cell wall- or membrane-related, transporter-related, stress-related, and signal transduction related (see Appendix). In this section, it is discussed how these genes could be functionally implicated in the global cellular response to glycine. The molecular architecture of the cell wall is not fixed but highly dynamic, and regulated depending on internal and external cues. For example, the cell can make considerable adjustments to the composition and structure of its cell wall during the cell cycle or in response to environmental conditions such as nutrient and oxygen availability, temperature, and pH (Kapteyn eta!., 1999). From studies of cell wall proteins, it is now clear that specific cell wall composition is determined by growth conditions as well as cell division cycle phase (Wodicka et al., 1997). Therefore, altered expression levels of genes that are related to the cell wall or membrane may result from changes in the medium composition and/or metabolic and biochemical changes within the cell. In most organisms including S. cerevisiae, transport of one-carbon units between mitochondria and the cytoplasm relies on one-carbon donors such as serine, glycine or formate because THF derivatives can not cross the mitochondrial membrane to any 161 Genome-wide transcript analysis significant extent (Cybulsky and Fisher, 1981; Horne et a/., 1989). However, the mitochondrial transport mechanisms for glycine and serine intercompartmental flow are not known. The investigation of these transport mechanisms is very interesting and important for the understanding of one-carbon metabolism. It will provide an insight into how the one carbon metabolic flow between the two compartments is controlled and communicated. It is likely that the mitochondrial glycine/serine transport systems may be regulated by the availability of glycine and serine within the cell. Comparison of genome-wide transcript profiles under different nutritional conditions may help to identify such proteins. Among those genes identified related to the major facilitator superfamily proteins (Nelissen et al., 1997) or transporters, there are several of unknown function. These are potential candidates for glycine and serine transporters. Further studies are required for the functionality of these proteins. Signal transduction-related genes were also identified. These may be needed for the cell to relay various extra- and intracellular signals for the integrated global response to the changing environment. It is evident that this gene-array analysis has identified genes that do not appear to be directly needed for the glycine response. These include the stress-related genes and other genes whose functions were not classified into distinct groups and for which no connection to one-carbon metabolism is yet apparent. These genes probably represent indirect effects of the glycine response. It is possible they are mediated by one or more intermediary factors and reflect an adaptive response rather than a direct consequence of the glycine response. In functional genomics, the activity of all an organism's genes can be examined. Since not all of them will be relevant to the property under study, it is necessary to define the physiological or developmental state of the cells very accurately. In this study, mRNA was prepared from two separate cultures (Dmin and Dmin with I 0 mM glycine) which were grown to reach an OD6oo of 0.5. In this case, there was ample time for the cells to adjust themselves to the given conditions. In other words, no distinction could be made between 162 Genome-wide transcript analysis the expression of genes not immediately related to the additional glycine and those that are primarily affected by glycine in the medium. To resolve which effects are a primary consequence of the given condition and which are secondary or indirect effects, further detailed analyses are required. One way to approach this issue will be to do a time-course experiment. This would make it possible to determine at what time the affected genes start to be expressed. For example, to search for genes that are primarily affected by the addition of 10 mM glycine in the medium, one can grow cells in minimal medium first and then transfer to fresh medium containing 10 mM glycine and performing transcript analysis at appropriate intervals. By measuring and comparing the transcript levels of genes that are known to be primarily affected by glycine addition such as the GCV genes, determination of the minimal time required to see the changes in expression levels can be made. Based on these results, it will be possible to discriminate which genes are primarily or secondarily expressed genes. The interconnectedness of cell pathways and the information of their effects on one another's gene expression is very valuable for the accurate interpretation of transcriptome data as well as for an understanding the relationships between them. 5.3 Future Direction. Results from the gene-array analysis by the m1m-array hybridisation method demonstrates that it is a powerful, effective and relatively simple tool for identification of genes that are transcriptionally controlled by a given condition. It is then possible to construct an idea of the global metabolic changes within the cell when adapting to its changing environment. As more is learned about the function of each gene in the yeast genome, this technology will become remarkably more powerful. However, at this stage some considerations should be made when analysing the results. In S. cerevisiae, many genes have homologous counterparts within the genome. Since gene-array analysis uses dot blots, some cross-hybridisation of multiple labelled eDNA species with a single target open reading frame may occur. This presents a risk of 163 Genome-wide transcript analysis misinterpretation of the data and the identification of unrelated genes is possible. Northern analysis is useful to confirm and judge whether identification of a gene through gene-array analysis is due to a cross-hybridisation event or an actual altered gene expression state. In addition, recent studies of the relationship between mRNA and protein expression levels have shown that mRNA levels do not necessarily correlate with protein levels (Gygi et al., 1999). The desired end-point for the functional description of a biological system is therefore the accurate measurement of protein expression levels and their respective activities. Nevertheless, the gene-array hybridization technique is an undoubtedly powerful tool to analyse gene expression on the genomic scale and gives a perspective on the complexity of changes that occur in the cell when responding to environmental stimuli. It also provides a foundation for many relevant areas of further research; in this system alone it has identified many genes whose function can now be investigated in more detail with the knowledge that they may be closely related to one-carbon metabolism 164 Conclusion Chapter 6: GENERAL DISCUSSION AND PERSPECTIVES In this chapter, the significance of this study is briefly described (section 6.1), followed by a general discussion on the transcriptional control mechanisms of dual functional transcription factors known in eukaryotes (section 6.2). Possible models for transcriptional regulation conferring the glycine response of the GCV genes are also presented (especially for that of the GCV2 gene). Finally, suggested future directions for this work are indicated. 6.1 One-Carbon Regulon The work presented in this thesis focussed on determining how eukaryotic cells regulate the synthesis of enzymes involved in one-carbon metabolism using the model organism, Saccharomyces cerevisiae. Many metabolic pathways are very similar between yeast and higher eukaryotes, and the general transcriptional control mechanisms found within eukaryotes are essentially identical. Therefore, this thesis provides a foundation for an understanding of how transcriptional control of one-carbon metabolism is achieved in eukaryotes. Numerous biochemical and genetic studies have been reported in eukaryotes (for review see Cossins and Chen, 1997) determining the fate of one-carbon units in the cell. However, there has been very little work on regulation of eukaryotic one-carbon metabolism at the molecular level (Slansky and Farnham, 1996). Although there are detailed studies on regulation of one-carbon metabolism in E. coli (Ghrist and Stauffer, 1998; Ghrist and Stauffer, 1995), the prokaryotic one-carbon metabolism and transcriptional control systems are qualitatively different from those of eukaryotic systems presented here. The most significant and important finding of this work is the identification of the "one-carbon regulon" in the transcriptional control of one-carbon metabolism genes in 5. cerevisiae. It has been identified that THF (or its derivative) is the mediator of the one carbon regulon (chapter 4). THF is the central molecule in one-carbon metabolism 165 Conclusion (section 1.2) and its involvement in the transcriptional control of GCV genes, together with the identification of one-carbon metabolic genes that have increased expression in the presence of glycine in the medium (section 5.2) indicates that genes involved in one carbon metabolism are co-regulated at the transcription level by the one-carbon regulon. Determination of the extent of this regulatory system and identification of other genes that are under this control (such as mitochondrial glycine/serine transporter encoding genes) are currently under way using time-course genome-wide analyses (section 5.2.6). , Another interesting finding of this work is that the glycine response of the GCV genes can be mediated by either activation or repression. To provide an insight in to regulatory mechanisms of the one-carbon metabolism genes, examples of transcriptional control mechanisms by eukaryotic dual function factors are outlined below. Section 6.3 describes possible models of GCV2 gene regulation based on the results obtained from the previous chapters. These models are not restricted to transcriptional regulation of GCV2, but also apply to the other GCV genes and to other genes which show a glycine response that are related to one-carbon metabolism. 6.2 Dual Acting Transcription Factors In eukaryotes, it is quite common for a transcription factor to serve either as an activator or as a repressor. It appears that some proteins have both activating and repressing potential, and the predominant effect depends on promoter context, or the presence of other factors including other gene regulatory proteins and ligands. The processes involved in the genetic switching of a transcription factor are various and seldom straightforward. The dual-functional transcription factors were shown to switch their activity by a range of mechanisms including the binding of a ligand, interaction with other regulatory proteins, cooperation with a neighbouring DNA-bound transcription factor, different locations of a binding site with respect to the transcriptional start site, concentration dependent homodimerisation, interaction with specific DNA sequences, and alterations in the structure of DNA or chromatin promoting DNA-protein or protein-protein interactions 166 Conclusion (Roberts and Green, 1995). Many regulatory elements of these kinds exert their proper activation or repression effect only when positioned within their natural DNA contexts (context-dependent regulation). In addition, most of the dual-functional regulators were shown to contain both activation and repression domains, indicating that the differential activity of such a transcription factor may result from an interplay between its independent functional regions. One or more of the above mechanisms may enable the glycine regulatory region (GRR)-binding protein(s) to either activate or repress transcription, depending on the DNA or physiological context in the cell. Figure 6.1 illustrates some of the mechanisms of activator-repressor switching. First, switching can be brought about by concentration-dependence (Figure 6.1A). The Drosophila zinc-finger protein, Kruppel has activation or repression activities solely dependent on its concentration. Monomeric Kruppel interacts with TFllB to activate transcription, whereas dimeric Kruppel which forms as a result of an increase in concentration, interacts with TFIIE to repress. Kruppel dimerisation presumably masks the TFIIB-binding surface and creates a TFIIE-binding site. This could result from a dimer-induced conformational change or possibly be formed by a contribution from each of the two Kruppel subunits (Sauer et al., 1995a; Sauer and Jackle, 1993). The activator/repressor effects of p53 also depend on the intracellular concentration of p53 per se. At lower concentrations, p53 activates transcription while high concentrations of p53 repress transcription (Kristjuhan and Maimets, 1995). However, it appears that this type of mechanism is not used for the glycine response of the G CV genes. If the activity of the transcription factor is altered in a concentration-dependent manner, the DNA-protein complexes formed would differ in repressing conditions (Dmin) compared with activating conditions (with the presence of glycine in the medium). From the DNA-protein interaction studies using nuclear extracts from cells grown under different nutritional conditions (Chapter 4), it was shown that the pattern of complex formation was unchanged regardless of changes to the growth conditions. This can be confirmed in vivo, once the ORR-binding protein is known; the 167 Conclusion gene that encodes this protein cloned into an expression vector will test whether this mechanism is involved in regulation of the GCV genes. Secondly, the switching can also occur through interactions with other regulatory proteins or with auxiliary factors (Figure 6.\B). For example, a mammalian zinc-finger protein, YY 1 typically acts as a repressor, but can be converted into a potent transcriptional activator in the presence of adenovirus E Ia protein (Figure 6.1B; top panel) (Shi eta!., 1991 ). In another case, different homo- or heterotypic interactions of bHLH-bZip class of proteins, Myc, Max and Mad transcription factors confers switching (Figure 6.1B; middle panel). It was suggested that the Max homodimer or Mad-Max heterodimer can act as repressor, and the Myc-Max heterodimer mediates activation (Ayer et al., 1993; Kretzner et al., 1992). In the other case, the product of the tumour suppressor gene, WTJ is modulated in its ability to transactivate or repress transcription through interaction with p53 (Figure 6.\B; bottom panel). The intrinsic property of WTJ is as an activator, however, physical association with p53 converts it to a repressor (Maheswaran eta!., 1993). An analogous switching mechanism (Figure 6.\B; bottom panel) can also be found inS. cerevisiae. Mig1p, a regulator involved in glucose repression, is an activator rather than a repressor in an ssn6 mutant or tupl mutant. However, when Mig1p is bound by a SSN6/TUP1 complex, it represses target gene transcription (Treite1 and Carlson, 1995). Tup1p and Ssn6p transcriptionally repress a wide variety of genes (regulated by glucose, cell type, oxygen, and DNA damage) in yeast but they do not appear to bind DNA, and it has been suggested that specific DNA-binding proteins recruit the SSN6/TUP1 complex to different promoters (Komachi et al., 1994; Tzamarias and Struhl, 1995). This switching mechanism requires multiple protein-protein interactions whereby additional proteins bind to the transcription factors and modulate their function. These accessory proteins and protein complexes may form the interface for the input of regulatory information to the transcriptional apparatus. Studies of in vitro DNA-protein interactions using heparin-Sepharose fractions showed that there are multiple complexes 168 Conclusion formed with the GRR. It is likely that there are multiple protein-protein interactions for the glycine response, and identifying these will be important for an understanding of the mechanism of transcriptional regulation. A two-hybrid system to study the protein- protein interactions (Fields and Song, 1989) may therefore prove fruitful for future studies. Activation Repression Binding TATA Binding TATA element element B c& <3) r CJ <3) r< Binding TATA Binding TATA element element 00 <3) r< [ X 1 <3) r c& <3) rx Figure 6.1 Mechanisms of activator-repressor switching. Each of the transcriptional regulators is shown in either an activation (left) or repression (right) context. Binding sites of the regulators and TATA boxes are shown. Different transcription factors or auxiliary factors are indicated as X, Y or Z. PIC indicates transcription preinitiation complex. A. Switching can be dependent upon concentration of the transcription factor. An increase in the concentration of transcription factor causes a switch from an activator to a repressor. B. Protein-protein interactions can facilitate the activator repressor switching. The top and bottom panels illustrate that other proteins (Y) can interact with the DNA-bound transcription factors (X) to result in the switching. The middle panel shows different combinations of homo- or hetcrodimer formation causes switching. 169 Conclusion Thirdly, switching may depend on the context of the binding sites for the transcription factors. YY I is a mammalian multifunctional transcription factor that can either activate or repress transcription depending on promoter architecture. YY 1 can either activate or repress the c-fos gene based on the orientation or the position of a YY1- binding site within the promoter (Natesan and Gilman, 1993). Another example is Sp3, an ubiquitously expressed dual-function regulator whose activity is also dependent upon promoter context. Sp3 represses transcription of promoters bearing multiple DNA binding sites, whereas it activates isogenic reporters containing a single binding site (Majello eta!., 1997). The yeast protein Rap 1p (repressor activator protein-]) is a sequence-specific multifunctional DNA-binding protein that is essential for cell viability. Rap1p contains distinct activation and silencing domains and can function as an activator or a repressor depending on the context of its binding site. In the MATa promoter, there is a Rap 1p binding site that mediates activation (Giesman et al., 1991). However silencing occurs when there are multiple binding sites for this protein, as at telomeres (Kyrion et al., 1993). This kind of mechanism does not appear to be functional in the regulation of the CCV genes since neither multiple copies of the GRR nor its orientation affected the nature (activation or repression) of the transcriptional regulation when the GRR was tested in heterologous promoters (section 3.3.1 and 3.3.2). A fourth mechanism of switching is achieved by binding of a ligand (Figure 6.1C). The steroid, retinoic acid and thyroid hormone receptors belong to a large family of proteins that function as ligand-activated transcription factors. The human thyroid hormone receptor-13 (hTR/3) is converted from a repressor to an activator upon binding of its ligand, thyroid hormone. The ligand-free hTR/3 has been shown to repress transcription independently of position and orientation, and repression is effective on a complete or on a minimal promoter (Baniahmad eta!., 1990). As a repressor, the transcriptional repression domain of hTR/3 interacts with TFIIB that is required for pre initiation complex formation (Baniahmad eta!., 1993). Therefore it appears that hTR/3 170 Conclusion interacts with TFIIB to block further assembly of the preinitiation complex. As an activator, the transcriptional activation domain of hTRB interacts with the basic region of TFIIB, the same part of TFIIB that is contacted by acidic activators (Roberts eta!., 1993). Thyroid hormone binds to the repressor region of hTRB and disrupts the interaction with the TFIIB amino terminus, thereby relieving repression (Baniahmad et a/., 1993). Activation Repression c Binding TATA Binding TATA element element D(X) ) QD r 0~ QD r< Binding TATA Binding TATA elements elements CXJ; I QD r (§) rx Figure 6.1 Mechanisms of activator-repressor switching. Each of the transcriptional regulators is shown in either an activation (left) or repression (right) context. Binding sites of the regulators and TATA box are shown. Different transcription factors are indicated as X, Y or Z. C. Switching can be caused by a ligand (black square). D. Transcriptional effects can be reversed by a neighbouring transcription factor (Y and Z) binding to distinct DNA elements. Upper panel shows that in the presence of neighbouring DNA-bound protein a regulator switches from an activator to a repressor. The lower panel illustrates that the same transcription factor (X) can be acting either as an activator or a repressor depending on the presence of different neighbouring DNA-bound transcription factors. 171 Conclusion A similar mechanism is operative for the Leu3p transcription factor of S. cerevisiae, which is a regulator of branched-chain amino acid biosynthesis (Brisco and Kohlhaw, 1990; Friden and Schimmel, 1988). It mediates activation only in the presence of a-isopropylmalate (a-IMP), a product of the first committed step in leucine biosynthesis. In the absence of a-IMP, Leu3p reduces basal level transcription four- to five-fold (Sze eta/., 1992). Binding of the modulator a-IMP causes conformational changes of Leu3p which eventually exposes its activation domain and thus strongly activates gene expression via interacting with components of the transcription apparatus (Wang eta/., 1997). From the investigations of the interactions between the ORR-binding protein and THF using electrophoretic mobility shift assays (EMSA) and gene expression studies with a mutant unable to synthesise THF (Chapter 4), this activator/repressor switching mechanism appears an attractive model for the regulation of the glycine response of the GCV genes; THF acts as a ligand for the ORR-binding transcription factor to regulate expression of the GCV genes. Section 6.3 outlines models that are relevant to a physiological context for GCV2 regulation involving THF (or a derivative) as a ligand. Finally, the transcriptional effect of a specific transcription factor can be reversed by a neighbouring DNA-bound protein (Figure 6.1D). The activators of the Drosophila Rei family can be converted to repressors by a cofactor designated DSP I, which binds to NRS sequences associated with Rei-binding sites in several cellular promoters (Figure 6.1D; upper panel) (Lehming et al., 1994). Human transcription factor YYI and yeast Raplp were previously discussed as switching their repressor/ activator activity depending on the context of their binding sites (the third mechanism of switching). These proteins can also switch their activity by interaction with a neighbouring DNA-bound protein. YY I can either repress or activate a certain promoter depending upon the presence of an adjacent DNA sequence that binds to a distinct nuclear factor (Bauknecht et al., !995). YYI binding appears to preclude the binding of activator proteins (Lu et al., 1994). The S. cerevisiae multifunctional transcription factor, Raplp plays a central role in the complex system of combinatorial controls, participating in different sets of protein- 172 Conclusion DNA and protein-protein interactions (Shore, 1994). For example, as illustrated in Figure 6.1 D (bottom panel), in promoters of genes encoding glycolytic enzymes that contain a Gcrlp-binding site near a Raplp binding site, Gcrlp and Raplp mediate activation of transcription (Tornow et ul., 1993). However, in the HM silent mating loci, the RAP site is flanked by an ACS (origin recognition complex-binding site) and mediates silencing (Bell and Stillman, 1992). In the regulation of GCV2 gene expression, extensive deletion analyses revealed that sequence 5' of the GRR (-310 to -289), plays a role in the full glycine response. Moreover, EMSA using heparin-Sepharose fractions showed a DNA-protein complex formed with the DNA fragment spanning from -322 to -295. This complex was separable from the other three complex forming activities found with the GCV2 sequence from -309 to -267, indicating that the protein responsible for this complex formation may be that required for the full glycine response of GCV2 gene. The details for the involvement of this protein in the regulation of GCV2 are not clear but it is possible that the binding of this protein to the DNA-element adjacent to the CATCN7CTTCTT motif confers a combinatorial effect giving the full glycine response. Further studies such as footprinting and protein-protein interaction experiments will help determining the exact binding site of this protein and its role in the regulation of GCV2. Other possible mechanisms for activator-repressor switching have also been suggested. A functional dissection of RFXl, which binds to the EP element of the hepatitis B virus, identified activation and repression domains with independent transcriptional activity. It was proposed that there may be a direct interaction between the activation and repression domains within RFX I and the particular array of these domains in RFX I may be designed to allow the mutual neutralisation of their activities and under certain conditions, this self-neutralisation is relieved to achieve its dual-functionality (Katan et ul., 1997). The activity of some multi-functional regulators such as the lymphoid-specific transcription factor, Oct-2a (Friedl and Matthias, 1996), and a ubiquitous transcription factor, Sp3 (Majello et al., 1997) is reported to be dependent upon their cellular context. 173 Conclusion Their transcriptional activity was observed in some cell lines but not others, suggesting their effects are dependent on the nature of cofactors present in a particular cellular background. Unravelling the switching mechanism described above involves analysis of complex protein-protein, protein-DNA and protein-ligand interactions and understanding the interplay of these interactions is vital to fully appreciate transcriptional regulation. The multifunctional transcription factors may activate or repress different target genes in response to a single inducing agent in order to fulfill several functions in a specific cell type. It is apparent that in some cases, a dual-functional transcription factor can work via multiple switching mechanisms (Rap I p, YY 1 and p53, for example). Likewise, it is also possible that the regulation of the GCV genes for the glycine response may involve multiple control mechanism;. 6.3 Mudd for the Regulation of GCV Genes From protein binding studies it was shown that there are four major complexes formed with the promoter of the GCV2 gene (-309 to -267). One of them (complex II) was distinct from the others on the basis of which heparin-Sepharose fractions contained the protein involved (Figure 4.1 0; complex II) and this complex was only formed when sequences between -322 to -295 (Figure 4.13). Therefore, complex II formation relies on sequence between -309 and -295. Furthermore, titration experiments with the heparin-Sepharose fraction containing complex I forming activity showed that this DNA binding protein can dimerise (Figure 4.14). This indicates that among the four complexes formed with the GRR, two of them possibly resulted from one DNA-binding protein. The identity of the remaining complex is not clear; this may be caused by a further interaction between two DNA binding proteins or be a proteolytic product of the higher order complex. Footprinting analysis of the GCV2 promoter with a heparin Sepharose fraction containing complex I activity showed that protection from DNasei activity covered the sequence, 5'-CATCN7CTTCTT-3' which was shown to be the GRR from gene analyses and alignment of the GCV genes (discussed in chapter 3). 174 Conclusion GCN4/BASI TAT A Box !:! : I I II I II I I I I • C: I -309 to -295 GRR N-element Inr Dmin ~------"' I L-----. GCN4/BAS 1 -----I Dmin +glycine ~-----' i ..... i I L - - - - - . GCN4/BAS 1 - - - - -I NitrogenQ nch I , . Ill II II •..... : Figure 6.2. Models for the transcriptional control of the GCV2 gene. The top panel of the diagram shows the positions of putative regulatory factor-binding sites, the TATA box and transcription initiation site (lnr) of the GCV2 promoter. Models for the transcriptional control under three different conditions are suggested. Arrows indicate transcriptional activation and bars indicates repression. The putative GRR and N-element-binding proteins are shown either as a circle or ovals and THF is represented by a filled square on the binding proteins. InN-rich conditions, the line with arrows at both ends indicates the potential interaction between the ORR and N-element. 175 Conclusion Interestingly, a GCV2 promoter sequence with mutations of the CTTCTT core motif within the ORR could still form all four complexes, albeit at a much reduced level. This was consistent with the result of site-directed mutagenesis showing that a CTTCTT mutation did not completely abolish the glycine response of the GCV2. It is possible that the protein bound to the region between -209 to -295 may stabilise the interaction between DNA and the ORR-binding protein. Therefore, the protein responsible for complex I formation is the probable transcription factor essential for the glycine response and the protein that binds to the region between -309 to -295 (complex II formation) may be necessary for the full glycine response by stabilising the ORR-binding transcription factor. Figure 6.1 illustrates the position of each element identified in this study and interactions of proteins with these elements and also provides a model for transcriptional control of the GCV2 gene under three different conditions: Dmin, Dmin supplemented with glycine and N-rich medium (rich nitrogen source medium or YEPD). The CCV genes are expressed to some extent on minimal medium, in this situation the cells would be forming serine via the glycolytic pathway and converting it to glycine via the serine hydroxymethyltransferase (SHMT). The SHMT reaction also converts THF into 5, !O-CH2-THF which is required for one-carbon metabolism. Glycine can be used by the GDC for the mitochondrial production of one-carbon metabolites. Under normal growth conditions (Dmin), the cytoplasmic concentration of THF is expected to be at a lower level compared to that in cells grown with added glycine since serine produced by the glycolytic pathway is the major catabolite for producing one carbon units and excess glycine would act as an end-product inhibitor of the conversion of serine to 5, I O-CH2-THF and glycine. It is proposed that in this situation, limiting THF causes the ORR-binding protein to act as a repressor (Figure 6.2). From genetic studies with various regulatory mutants (section 3.4), the basal transcriptional level of GCV2 has been shown to be controlled by other transcription factors such as Gcn4p or Bas I p, and the nitrogen regulatory element. In the absence of good nitrogen sources such as glutamine or asparagine, cells can use the GDC reaction 176 Conclusion for the production of a nitrogen source and therefore repression through the nitrogen regulatory element would not be operative. It was also shown that Ocn4p and Bas1p are involved in the regulation of the GCV2 gene, since a gcn4, basi double mutant showed a significantly reduced level of GCV2 expression in both Dmin and Dmin with glycine (section 3.4.1). Because strains lacking these proteins still exhibited a glycine response, Ocn4p and Bas1p are not directly related to this control but instead are possibly involved in setting basal level expression. In the presence of excess glycine (when the glycine response is observed), the cell would be able to spare serine, and to obtain nitrogen from glycine in the absence of more readily assimilated nitrogen sources. The excess glycine in the cytoplasm of the cell would inhibit, as an end product, the serine-to-glycine conversion via SHMT, and if so this would reduce the generation of 5, IO-CH2-THF leading to higher levels of uncharged THF. THF acts as a ligand for the ORR-binding proteins converting them into activators while also increasing their affinity for the ORR (Figure 6.2). This could act as a cue to increase the activity of the mitochondrial glycine decarboxylase activity to generate serine from glycine and thus increase the flux of one-carbon metabolites. Under rich nitrogen growth conditions (or in YEPD), the predominant transcriptional influence is repression through the nitrogen-regulatory element. Moreover, the data suggest that there is an interaction between the ORR and the nitrogen regulatory element (section 3.4.2). An alternative model for the transcriptional regulation through the ORR is possible, in which the effector molecule is not THF, but a related derivative. In section 4.1.3, it was shown that the concentration of THF effecting the in vitro binding of protein to the ORR was above the normal physiological level. This could be due to the use of monoglutamyl THF instead of polyglutamyl THF which is the physiologically active form of THF (section 1.2.1 ), but it is also possible that THF is not the actual molecule to function as a ligand. S,lO-CH2-THF is a good candidate for an effector molecule since it occupies a critical position in one-carbon metabolism from which various pathways 177 Conclusion diverge. Therefore, the cellular level of 5,IO-CH2-THF would serve as a good indicator for intracellular one-carbon metabolic status. It is proposed that the binding of 5,lO-CH2-THF to the glycine regulatory protein functions in the opposite way from that proposed for THF; its binding increasing the activity of the protein bound to the GRR as a repressor. As discussed in the previous model system, the cellular concentration of 5, IO-CH2-THF will be relatively higher in cells grown in Dmin than in Dmin with glycine. In this situation, 5,l0-CH2-THF would bind to the GRR-binding protein and repress GCV2 gene expression. Conversely, when the 5,10-CH2-THF to THF ratios are lower (cells grown in Dmin with glycine), the ligand-free regulator may function as an activator. Both model systems also cater for why expression of the GCV gene was not affected by the addition of serine (Figure 3.3). Addition of serine would cause a decrease in the level ofTHF along with an increase in the level of 5,10-CH2-THF, and therefore maintain repression of the GCV genes. However, the result of the gene-array analysis indicated that the addition of glycine to the medium would probably increase the cellular level of serine. It has been proposed that this occurs by glycine being channelled through the GDC and mitochondrial SHMT to produce serine which is utilised for cytoplasmic one-carbon metabolism (McNeil eta/., 1996). This raises an interesting point because if THF or 5,lO-CH2-THF is the effector molecule, the increase in the cellular level of serine would also cause a decrease in the level of THF (or increase in 5,10-CH2-THF), thus activation of the GCV genes would be diminished. One possible explanation for this is that the pools of serine or THF/5, 1O-CH2- THF may be compartmentalised within the cell. There are numerous studies suggesting that multiple pools of one-carbon metabolites exist in the cell (section 1.2.4). On this hypothesis, not all serine generated from glycine would be directed towards the production of 5,10-CH2-THF at the expense ofTHF, but some would accumulate within the cell. To support this claim, it has been shown that multiple pools of serine do exist when cells were grown with glycine as the sole one-carbon donor (Pasternack et al., 1994a). Furthermore, another study has shown that there are multiple pools of glycine in 178 Conclusion the mitochondrion, only one of which was available for mitochondrial serine synthesis (Pasternack et al., 1992). When cells are grown in the presence of excess glycine, it is possible that serine produced from glycine in mitochondria is separated in to a different pool from the serine that is utilised by cytoplasmic SHMT, thereby this will not affect the cytoplasmic levels of THF or 5,10-CH2·THF which determine the transcription of CCV genes. 6.4 The GRR of Other One-Carbon Metabolism-Related Genes The preliminary gene-array analysis presented here has provided a great deal of valuable information on the changes of the transcriptome in response to the addition of glycine to the medium. However, it became clear that additional analyses are required to clarify which genes responded as a result of primary effect of glycine addition. Nevertheless, those genes with altered expression related to one-carbon metabolism are excellent candidates for the primary effect of glycine addition. Based on the results of the gene-array analysis aml the GRR of the CCV genes revealed in Chapter 3 and 4, a potential consensus GRR for these genes was sought (Figure 6.2). Most of these genes contain similar sequence element(s) within their promoter regions. Gene potential GRRs position CCV2 CATC tctgact CTTCTT -297 CCVI CATC gccgtga CTTCTT -188 GRR CATC N? CTTCTT CCV3 CATg tgtaggg CTTCCTT -182 SHM2 CAgt gtgggcag CTTCTT -518 gAaC catgagt CTTCTT -153 DFRJ CATC ctatgta CTTCTT -377 MTDJ CtTg caaccag CTTCTT -125 CAaC cagctt CTTCTT -121 Figure 6.2. The potential GRR of genes related to one-carbon metabolism. A search for potential GRRs within the promoters of genes that showed increased expression in minimal medium containing glycine is shown above. The putative GRR of the CCV I and GCV2 genes served as a consensus sequence for the search in other genes' promoters. Capital letters indicate matches with the putative GRR. The number of intervening nucleotides between CATC and CTTCTT varies from 6 to 8 to allow for better alignment. 179 Conclusion Subsequent analyses are required to verify if these genes are glycine responsive and if the putative elements are functional in their respective promoters. By determining which are the primarily expressed genes when glycine is in the medium (section 5.2.6), a better consensus sequence of the GRR can be resolved, which should further help identification of genes commonly controlled by this one-carbon specific control system. 6.5 Future Directions One of the aims of this project was to identify all yeast genes that are co-regulated by one-carbon metabolism. It also aimed to identify the signal molecule conveying information of the physiological status of the cell to the transcriptional machinery for the efficient expression of enzymes required for the cell to adapt to the changing environment. It was also suggested that direct binding of THF or a derivative to the transcription factor(s) could modulate its activity. Studying the molecular basis of the ligand binding to the transcription factor(s) and interaction with the DNA element will further the understanding of control of the one-carbon metabolism in the cell. Therefore, future studies should include identification of the signal molecule for transcriptional regulation of one-carbon metabolism. This could be achieved in vivo by using various one-carbon metabolic mutants, or in vitro with EMSA using various THF species and polyglutamylated derivatives. Another interesting area of study is to elucidate how the GRR cis-element can function as an activator in one context and as a repressor in another. Detailed biochemical and genetic analyses will provide an insight into these regulatory aspects. The purification and N-terminal or internal sequencing of the GRR-binding protein is under way using various chromatographic steps including DNA-affinity chromatography immobilised GRR of the GCV2 gene (M. Piper, UNSW). The full gene encoding the transcription factor will then be identified from the yeast genome database and cloned. Once identified, the gene encoding the transcription factor can be cloned into an appropriate expression vector and produced for use in more detailed biochemical analyses such as the kinetics of ligand and GRR binding. It would also be possible to dissect the protein into the domains that are responsible for DNA-binding, ligand binding, 180 Conclusion activation/repression, dimerization, nuclear transport, and post-transcriptional modifications if these apply. It will also allow identification other factors that interact with this protein using the two-hybrid system (Fields and Song, 1989). Furthermore, generating a mutant lacking this protein will determine whether it is essential or necessary under a range of growth conditions. This mutant also can be used for gene-array analysis to investigate changes in global gene expression profiles. 181 References Abastado, J.-P., Miller, P. F., Jackson, B. M., and Hinnebusch, A. G. 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(1991) Activation of the erythropoietin receptor promoter by transcription factor GAT A-1. Proc. Nat!. Acad. Sci. USA 88: 10638-10641. 213 APPENDIX: Genes identified in the miniarray membrane hydridisation Unknown function (39) Fold! Gene Function 47.9 YNL019C Protein of unknown function. 8.9 YDR383C Protein of unknown function. 5.6 YAR053W Protein of unknown function (has 17% serine and threonine; has 2 potential membrane spanning regions). 5.2 YDR315C Protein of unknown function. 3.8 YPL099C Protein of unknown function. 3.6 YLR173W Protein of unknown function. 3.4 YDL086W Protein of unknown function. 3.4 YML066C Protein of unknown function. 2.9 YHR083W Protein of unknown function. 2.9 YBL062W Protein of unknown function, probable non-coding ORF. 2.8 YFL023W Protein of unknown function (rich in glutamic acid; similar to GIM5-tubulin biosynthesis; mutant is osmosensitivc). 2.6 YER050C Protein of unknown function. 2.6 YMLI08W Protein of unknown function. 2.4 YAL045C Protein of unknown function. 2.4 YJL202C Protein of unknown function. 2.3 YDR133C Prolcin of unknown function. 2.3 YJLI78C Protein of unknown function. 2.2 YDL096C Protein of unknown function. 1. Fold change from Dmin to Dmin with glycine. Decreased expression levels were shown as minus (-)figures. Genes that were identified visually for which quantitation was not possible are indicated as either R (increased expression) orB (decreased expression). 214 2.1 YEL033W Protein of unknown function .. 2.1 YOLIOIC Protein of unknown function (has similarity toY dr491 p;has prokaryotic lipoprotein lipid attachment site and to Yo1002p;oliate response element in the upstream). 2.1 YBRI13W Protein of unknown function. 2.0 YBRI97C Protein of unknown function. 2.0 YGROI8C Protein of unknown function. 2.0 YDR015C Protein of unknown function. (R) YGL015C Protein of unknown function. (R) YDR319C Protein of unknown function. (R) YPLI84C Protein of unknown function (has putative RNA binding region). (R) YKR007W Protein of unknown function. (R) YMR099C Protein of unknown function. (R) YJRI46W Protein of unknown function. (R) YLR414C Protein of unknown function (has prokaryotic lipoprotein lipid attachment site). (R) YLRI69W Protein of unknown function. -92.7 YOR364W Protein of unknown function. -7.2 YNU13C Protein of unknown function (has similarity to ORf T20B 121 of C. elegans). -2.6 YGRI65W Protein of unknown function. -2.0 YGRI34W Protein of unknown function (has similarity to C. elegans protein of unknown function on cosmid C27H5). (B) YOLI41W Protein of unknown function, similarity toY dd35p. (B) YLRI56W Protein of unknown function. (B) YKL206C Protein of unknown function. 215 One-carbon metabolism and related. (13) 13.0 YERI83C Protein with similarity to human 5, I 0-methenyltctrahydrofolatc synthetase. 11.0 YPLI76C Protein with similarity to Ssp 134p. Similarity to H. sapiens and C. elegans folate hydrolase. 3.5 SHM2 Serine hydroxymethyltransfcrase (glycine hydroxymcthyltransferase), intcrconverts serine and glycine. 3.3 MTDI NAD-depcndent 5, 10-methylenetetrahydrafolate dehydrogenase. 2.9 ADEI3 Adenylosuccinatc lyase, carries out the eighth step in de novo purine biosynthcsis;convcrts phosphorihosylaminoimidazolc succinocarboxamide (SAICAR) to phosphoribosylaminoimidazolc carboxamide (AICAR). 2.6 GCVI Glycine decarboxylase T subunit (glycine cleavage T protein), functions in the pathway for glycine degradation. 2.2 GCV3 Hydrogen carrier protein; H-protcin subunit of the glycine cleavage system. (R) DFRI Dihydrofolate reductase. (R) GCV2 Glycine decarboxylase; pyridoxal phosphate containing subunit. (R) APT! Adenine phosphoribosyltrans[crase (APRT). (R) SAM I S-adenosylmcthioninc synthetase 1. (R) OPB/PEM2 Phospholipid-N-methyltransh:rase; carries out second and third methylation steps of the phosphatidylcholine biosynthesis pathway. (R) SPE4 Spermine synthase that synthesizes the final product in the pathway of polyamine biosynthesis. Catalyzes the condensation of spennidine. with dccarboxylated S-adcnosylmethioninc to form spermine. Has an SAM binding motif Amino acid metabolism (5) 2.6 ARGI Argininosuccinate synthetase (citrulline--aspartate ligase); catalyzes the penultimate step in arginine synthesis. 2.3 THRI Homoscrine kinase (ATP:L-homoscrine-0-P-transferasc); first step in the threonine biosynthesis pathway. 2.2 ASP3 L-asparaginase IL 2.1 LYS21 Homocitrate synthase; involved in lysine metabolism. 2.1 ARG3 Ornithine carbamyhransfcrase, catalyzes the sixth step in the arginine biosynthesis pathway. 216 Other metabolism (4) 5.9 ICLl Isocitrate lyase, peroxisomal, carries out part of the glyoxylate cycle, required for gluconeogenesis. 2.2 YIL042C Protein with similarity to mitochondrial branched chain alpha-ketoacid and pyruvate dehydrogenase protein kinascs. (R) C!Tl/GLU3 Citrate synthase, mitochondrial, converts acetyi-CoA and oxaloacctatc into citrate plus CoA. (R) PDBI Pyruvate dehydrogenase complex, El-bcta subunit. Transcriution (20) 5.1 TFB2/LPH5 Component or RNA polymerase transcription initiation TFIJH ([actor b); 55 kDa subunit. 3.7 M1G2 Zinc-finger protein involved in glucose repression of SUC2. 3.1 YPL146C Protein of unknown function (sequence contains heptad repeats of the leucine-zipper pattern). 3.0 GALli Component of RNA polymerase holoenzyme (mediator complex) with positive and negative effects on transcription of individual genes. 2.9 HAC! Transcription factor that activates the unfolded protein response pathway; mRNA splicing is regulated by Ire I p and only the product of spliced mRNA is able to induce the response. 2.4 MCMl Transcription factor of the MADS (Mcm I p, Agamous, Deficiens, SRF) box family, recruits coregulatory proteins for both gene activation and repression at a variety of loci. 2.4 ZDSl/NRCl Protein that regulates SWE I and CLN2 transcription. 2.1 IXRI lntrastrand crosslink recognition protein; has 2 HMO boxes and transcription factor that confers oxygen (02) regulation on COX5B. (R) SPT15 TAT A-binding component of RNA polymerase transcription initiation factor TFIID, component of RNA polymcra')e Ill transcription factor TFillB. (R) CTHl Protein of the inducible CCCH zinc finger family. (R) YBR150C Protein with similarity to transcription factors, has Zn[2l-Cysl61 fungal-type binuclear cluster domain in theN-terminal region. 217 (R) POL3/CDC2 DNA polymerase delta large subunit. (R) POP2/CAFI Component of the CCR4 complex. required for glucose derepression. (R) ABF2 Abundant mitochondrial DNA-binding protein with two HMG-box domains, required for maintenance, transmission and recombination of mitochondrial genome. (R) POL32 Small subunit of DNA polymerase delta. (R) CTH2 Protein of the inducible CCCH zinc finger family. (R) YOR267C Serine/threonine protein kinase with similarity to members of the NPR 1 (nitrogen permease reactivator; promotes activity of several penneasc under nitrogen dereprcssing conditions; repressed in the presence of preferred N-sourccs) subfamily. High serine content. (B) YMRI12C Component of RNA polymerase II mediator (SRB) suhcomplex. (B) YOR137C Protein of unknown function (has 1cucinc-zipper motit). (B) RPAI4 RNA polymerase I subunit A 14. F-box containing proteins (4) (B) CDC4 F~box protein and component of the SCF~Cdc4p complex (Skp I p~Cdc53p~Cdc34p~Cdc4p) which targets Sic I p, Far I p, Cdc6p, Ctfl3p and Gcn4p for ubiquitin-dcpendent degradation, has WD (WD-40) repeats. N-terminal domain rich in serine, alanine, asparagine and thereonine. (R) CTFI3 CTF 13/CBF3C Component (subunit c) of Cbl] kinetochore complex; contains an F~box domain. (R) YLR097C Protein of unknown function, contains an F-box; originally found in mammalian eye lin F, shared with MctJOp, Cdc4p, Grrlp, CtfiJ and 13 proteins of unknown function). (B) YLR224W Protein of unknown function, contains an F-box. 218 Cell wall or membrane (17) 3.1 YMR251W Protein has 63% identity to Ecm4p (for cell wall maintenance) over 369 amino acids. 2.8 YNR065C Protein with similarity to Peplp (vacuolar sorting receptor), Vthlp and Vth2p. 2.7 GPI2 Protein involved in synthesis of N-acctylglucosaminyl phosphatidy1inosito1. 2.7 PMT2 Mannosyltransfcrase; (dolichyl phosphatc-0-mannose:protcin 0-D-mannosyhransferasc), involved in initiation of 0-glycosylation. 2.2 SED! Abundant cell surface glycoprotein that may contribute to cell wall integrity and protection from oxidative stress resistance; ovcrexprcssion suppresses growth defect of erd2. 2.0 YHR204W Protein with similarity to alpha-mannosidase and other glycosyl hydrolases. (R) YCKI Casein kinase 1 isoform. Associated with plasma membrane. (R) YBRI87W Protein with similarity to ND5 and PSB2. Contains 6 potential transmembrane segments. (R) SNC2 Synaptobrevin (v-SNARE) homolog present on post-Golgi vesicles. Localised to the ER and cytoplasmically oriented type 2 integral membrane protein of secretory pathway vesicles. (R) ERV25 Component of COPII-coatcd vesicles. Has a potent transmembrane segment. (R) HOC! Subunit of the Anp 1p-Hoc lp-Mnn llp-Mnn9p mannosyltransferase membrane complex of the Golgi involved in cell wall integrity. (R) MCRI NADH-cytochrome b5 reductase. Similar to Yml125p. Mitochondrial targeting ( 1/3 molecules become anchored in the milo. outer membrane. Promoter contains putative STREs. (R) VPS24 Protein involved in sorting of proteins in pre-vacuolar endosome. Probable component of endosomal coat. (R) YBRI47W Protein has 7 potential transmembrane domains. (R) YLL023C Predicted integral membrane protein with two potential transmembrane segments. (B) CYP5 Cyclophilin of the endoplasmic reticulum, has a HDEL sequence for retention in the ER. Involved in the secretary pathway. (B) ERG3 C-5 sterol desaturase; an iron, non-heme, oxygen-requiring enzyme of the ergosterol biosynthesis pathway. Contains several transmembrane domains. 219 Trans~orter (11} 6.9 STSI Protein that when overcxpressed restores protein transport and rRNA stability to a sec23 mutation. 5.5 YGL057C Protein of unknown function (has similarity to Ptm I p). 2.4 SEC26 Coatomcr (COPI) complex beta chain (beta-COP) of secretory pathway vesicles; required for retrograde transport from Golgi to endoplasmic reticulum. 2.3 HXT16 Protein with similarity to sugar transport proteins. 2.1 JENI Protein with similarity to E. coli osmoregulatory proP proline/betaine and KgtP alpha-ketoglutarate transporters; member of the major facilitator superfamily 2.0 PHOSS Membrane protein involved in inorganic phosphate transport. 2.0 YJROOIW Protein of unknown function, member of subfamily I of the major facilitator superfamily (MFS-permeases/ membrane plurispanncrs), which includes 7 prott::ins (all are with unknown function inS. cerevisiae). (R) NHXI Late endosomal (mitochondrial) Na+fH+ antiporter. Member of major facilitator family. (R) PTMI Protein with strong similarity to Yhl017p, member of the major facilitator superfamily (MFS ). has a potential signal sequence and 6 potential transmembrane domains. -2.1986 HXTI Low-affinity hexose transporter; member of sugar permease family and induced by glucose only at high concentration. -2.0164 RUD3 Protein that relieves transport defect of uso 1- I mutant. PAUl family (4} 144.0 PAUS Protein with similarity to members of the PAUl family. 3.5 CWP2 Mannoprotein of the cell wall; member of the PAU I family. 2.8 YIL176C Protein with similarity to members of the PAUl family. (R) CWPI Mannoprotein of the cell wall; member of the PAUl family. 220 Stress (11) 4.0 SNZl Stationary phase protein and member of the stationary phase-induced gene family which includes Snz2p and Snz3p. 3.8 RVS161 Protein required for viability after N, C, or S starvation, for internalization step of endocytosis, and for cell fusion during mating; roles in endocytosis and in cell fusion are independent of one another. 3.1 HSPI2 Heat shock protein of 12kDa, induced by heat, osmostress, oxidative stress and stationary phase. 2.0 TSAl Thiol-specific antioxidant, abundant protein that protects against sulfur-containing radicals. (R) TTRI Glutaredoxin (thioltransferase) (glutathione reductase). (R) TRXI Thiorcdoxin I. (R) DDR48 Stress protein induced by heat shock, DNA damage, or osmotic stress. (R) DJN7 Product of DNA damage inducible gene. (R) PRY2 Protein expressed under starvation conditions. -5.0 ATX2 AnTi-Oxidant 2, Manganese-trafficking protein. (B) YGPI Secreted glycoprotein produced in response to nutrient limitation. Si~:nal transduction (10) 13.3 YPT52 GTP-hinding protein of the rab family (ras supertllmily) involved in endocytosis and transport of proteins to the vacuole. 2.5 BMH2 Homolog of mammalian 14-3-3 protein, has strong similarity (93o/c} identity) to Bmh 1p, required for RAS/MAPK cascade signalling. 2.4 CAKI Cdk-activating kinase (serine/threonine protein kinase) responsible for ;n vivo activation of Cdc28p, also involved in spore wall formation. 2.3 YMR097C Protein of unknown runction. has ATP/GTP-binding site motif A (P-loop). (R) GSP2 GTP-binding protein member of the ras superfamily; involved in trafficking through nuclear pores. (R) YPT7/AST4 GTP-binding protein of the rab family (ras superfamily) with a role in protein transport between endosome-like compartments. 221 -2.8 YPL141C Serine/threonine protein kinase with similarity to Kin4p. -2.1 SDC25 GDP/GTP exchange factor for Ras. (B) YPT53 GTP-binding protein of the rab family; member of ras superfamily, involved in endocytosis and transport of proteins to the vacuole. (B) SCD25/SDC25 GDP/GTP exchange factor for Ras. Protein S)'nthesis {14) 12.1 VAS! Valyl-tRNA synthetase (mitochondrial and cytopla"mic forms are coded from the same gene), member of class I family of aminoacyl tRNA synthetases. 3.4 MRPLX Mitochondrial ribosomal protein of the large subunit (YmLS). 2.8 YGR146C Protein has 86% identity to Rp132p (for protein synthesis) from Klu.vveromyces lactis; 16% serine and 15% leucine. 2.3 HYP2/TIF51 A Translation initiation factor eiFSA; contains essential hypusinc modification. 2.2 MRPS28 Mitochondrial ribosomal protein of the small subunit. 2.1 TIFII Translation initiation factor elF I A. 2.1 RPL9B Ribosomal protein L9B. (R) SUII/RFRI 16 kDa translation initiation factor involved in initiation and in monitoring translational accuracy during elongation. (R) MPTI Protein required for protein synthesis. Interact with Prp9p (pre mRNA processing) in 2-hybrid system. (R) YNL247W Cysteinyl-tRNA synthetase. (R) MRP49 Mitochondrial ribosomal protein of the large subunit. -4.8 YNL284C Mitochondrial ribosomal protein of the large subunit (YmL I 0), member of the L 15 family of prokaryotic ribosomal protein. (B) RPLIA/SSMIA Large suhunit rihosomal protein L I A; (Rat LIOA) (Euhacterial L1) (Archcal L1 ). (B) MRPLIO Mitochondrial ribosomal protein of the large subunit (Y mL I 0), member of the L 15 family of prokaryotic ribosomal proteins. 222 Peptidase (3) 15.8 LAP3/GAL6 Aminopeptidase of cysteine protease family, homologous to rabbit bleomycin hydrolase. 7.2 CPS I Gly-X carboxypeptidase yscS, involved in nitrogen metabolism. 2.2 DOA4 Ubiquitin-spccific protease (ubiquitin C-tcrminal hydrolase) of the 26S proteasome complex; involved in vacuole biogenesis and osmoregulation. Others (36) 15.9 HOP2 Protein required for pairing of homologous chromosomes in meiosis. 5.6 HEM15 Fcrrochelatase (protoheme ferrolyasc), last step in heme biosynthesis pathway, catalyzes insertion of ferrous iron into protoporphyrin IX. 4.2 HST3 Protein with similarity to Sir2p (54% identity). 3.9 KEL2 Protein involved in cell fusion and morphology; contains six Kelch repeats. suspected mediating binding interactions with actin filaments. 3.3 BPHI Probable acetic acid export pump; has WD (WD-40) repeats in the C-terminal domain. 2.8 YDL228C Protein with similarity to Ach(ra klebsiana glutamate dehydrogenase. 2.8 HRTI Similarity to C. elegans protein of unknown function on cosmid F35G 12, overproduction reduces Ty3 transposition. 2.6 MMDI Protein important for Maintenance of Mitochondriall2I';A. 2.5 YDRI34C Probably pseudogene ORF, has strong similarity to FLO I. 2.5 SMEI Component of Ul and U2 snRNPs of the Sm class; required for mRNA splicing. 2.3 NAB3 Nuclear polyadenylated RNA-binding protein with I RNA recognition (RRM) domain, required for splicing .. 2.1 HRBI Protein with similarity to Rlf6p, has 3 RNA recognition (RRM) domains. localised predominantly to nucleus. 2.0 SUN2/RPN3 Non-ATPase component of26S protcasome complex. 2.0 STF2 ATPase stabilizing factor, binds to FO-ATPase; facilitates binding of inhibitor and 9 kDa protein to F 1-ATPase. (R) LIFI Protein that interacts with DNA ligase protein Dnl4p. 223 (R) ADH4 Alcohol dehydrogenase TV. (R) CAP2 Actin-capping protein, beta subunit. (R) QCR9 Ubiquinol cytochromc-c reductase subunit 9 (7.3 kDa protein); component of ubiquinol cytochrome-c reductase complex. (R) QCR7 Ubiquinol cytochrome-c reductase subunit 7 ( 14 kDa protein), component of uhiquinol cytochrorne-c reductase complex. (R) RIB3 DBP synthase; (3,4-dihydroxy-2-butanone 4-phosphatc synthase), part of the riboflavin biosynthesis pathway. (R) YCR063W Protein with similarity to Xenopus G I 0, a developmentally-regulated protein that is thought to he involved in translation during oocyte maturation. (R) ENTl Bpsin homolog required for endocytosis (R) HSTl Protein with similarity to Sir2p (72% identity). (R) YNL266W Protein with weak similarity to NADH dchydrogenases. weak similarity to a group of cytochrome c oxidases. (R) DBP6 Putative RNA helicase required for viability. (R) PFYl Profilin, can act to prevent actin polymerization; and to complex with monomeric actin. (R) YML125C Protein with similarity to NADH-cytochromc h5 reduc1ase. Sorted into 2 mitochondrial compartments. Contains putative STREs. (R) ERO! Protein required for protein disulfide bond formation in the endoplasmic reticulum. (R) DAKl Putative dihydroxyacetone kinase. 3-fold increase under hypcrosmotic stress. -3.1 YML059C Protein of unknown function (has 33% identity to human neuropathy target esterase protein over 850 amino acids). -2.7 YGL23oC Protein with strong similarity to E. coli gidA (Glucose Inhibited Division Protein A). -2.2 SRP54 Signal recognition particle subunit. (B) NUP2 Nuclear pore protein (nuclcoporin) with XFXfG motifs; has functional overlap with other proteins of nuclear pore complex. (B) HOS2 Protein with similarity to Hdalp, Rpd3p, Hoslp, and Hos3p. (B) AGA2 a-Agglutinin binding subuniL (30% serine and threonine). (B) FAS! Fatty-acyl-CoA synthase, beta chain; contains acetyl transferase, enoyl reductase, dehydratase, and malonyl/palmi toy! transferase activities. 224