THE MECHANISM OF ACTION OF INHIBITORY DOMAIN 1 OF GAL4
by CLAUDIA PERELLI HENTSCHEL
B.Sc.(H.) (Biochemistry), University of British Columbia, 1993
A THESIS SUBMITTED LN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE
THE FACULTY OF GRADUATE STUDIES
(Department of Biochemistry and Molecular Biology)
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THE UNIVERSITY OF BRITISH COLUMBIA
September, 1995
© Claudia Perelli Hentschel In presenting this thesis in partial fulfilment of the requirements for an advanced
degree at the University of British Columbia, I agree that the Library shall make it
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copying of this thesis for scholarly purposes may be granted by the head of my
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Department of
The University of British Columbia Vancouver, Canada
Date QMM*_ i0_
DE-6 (2/88) ABSTRACT
The mechanisms involved in the regulation of gene expression are of major interest in molecular biological research. Simpler models of lower eukaryotes such as yeast have been classically employed in such studies as a basis for the further characterization of more complex organisms. The galactose catabolism pathway of Saccharomyces cerevisiae provides an ideal model for research on gene regulation.
The carbon source available to the yeast dictates the expression of the GAL genes involved in the utilization of galactose in budding yeast. The key regulator in this pathway is the transcriptional activator GAL4. GAL4 is active in the presence of galactose and absence of glucose, the preferred carbon source. When glucose is present, though, GAL4 is rendered inactive by several glucose repression mechanisms. One of these mechanisms acts through the central region of the GAL4 protein itself, which is composed of a glucose-responsive domain and at least three inhibitory domains, one being inhibitory domain 1. The activity of the inhibitory domains has been suggested to be governed by the glucose-responsive domain.
With biochemical evidence, I suggest that the mechanism of transcriptional inactivation by inhibitory domain 1 occurs through multimerization of the GAL4 protein. Multimerization disrupts the formation of GAL4 dimers, preventing the transcriptional activator from binding to DNA and rendering the protein inactive.
ii TABLE OF CONTENTS
Abstract ii
List of Tables vi
List of Figures vii
List of Abbreviations ix
Acknowledgements x
INTRODUCTION 1
1. Eukaryotic Gene Regulation 1
2. Galactose Catabolism in S. cerevisiae 3
3. Induction by Galactose 4
4. Repression by Glucose 6
5. GAL4 Structure and Function 10
a. DNA Binding 10
b. Transcriptional Activation 13
c. GAL80 Interaction 14
d. GAL4 Phosphorylation 15
e. GAL4 Central Region 15
iii I
6. Aim of Research 17
MATERIALS AND METHODS 18
1. Ribonuclease Protection Assay 18
2. (3-Galactosidase Assays 19
3. DNA Binding Assays 19
4. Chemical Crosslinking Experiments 22
5. Experiments with E. coli Histidine-tagged GAL4 Derivatives 23
RESULTS 26
1. Glucose Inhibits GAL4 Through Its Central Region 26
2. ID1 Is a Strong Inhibitory Domain in Yeast 28
3. ID1 Impairs DNA Binding 31
4. ID1 Prevents Dimerization 42
5. ID1 Promotes Multimerization 46
6. ID1 Appears to Act by Itself 54
7. ID1 is Homologous to Other Proteins 66
DISCUSSSION 71
1. Glucose Inhibits GAL4 Through Its Central Region 71
i v 2. ID1 Is a Strong Inhibitory Domain Homologous to Other Proteins 72
3. ID1 Impairs DNA Binding 73
4. ID1 Prevents Dimerization and Promotes Multimerization 74
5. ID1 Does Not Require Other Cellular Factors 76
6. Comparison of ID1 to Other Known Inhibitory Domains 76
7. Model of the Mechanism of Action of ID1 77
REFERENCES 80
v LIST OF TABLES
Table 1. fi-Galactosidase Assay of GAL4 Derivatives 30
Table 2. Proteins Homologous to ID1 of GAL4 68
vi LIST OF FIGURES
Figure 1. Model of Transcriptional Activation 2
Figure 2. Galactose Catabolism Pathway in S. cerevisiae 5
Figure 3. Mechanisms of Glucose Repression 7
Figure 4. Functional Domains of GAL4 and Their Positioning 11
Figure 5. Glucose Represses GAL4 Through Its Central Region 27
Figure 6. GAL4 Derivatives 29
Figure 7. Expression of GAL4 Derivatives in E. coli 32
Figure 8. Expression of GAL4 Derivatives in Wheat Germ
Extracts 33
Figure 9. ID1 Impairs DNA Binding in E.coli in EMS A 35
Figure 10. ID1 Impairs DNA Binding of In Vitro Translated GAL4 in EMSA 37
Figure 11. ID1 Prevents DNA Binding in E. coli to a GAL4 Biotinylated Oligo 40
Figure 12. ID1 Prevents DNA Binding in Wheat Germ Extracts to a GAL4 Biotinylated Oligo 41
Figure 13. ID1 Prevents the Formation of Homodimers 43
Figure 14. In Vitro Co-translation of GAL4 Derivatives in Wheat Germ Extracts 45
vii Figure 15. ID1 Prevents the Formation of Heterodimers
Figure 16. Limited Crosslinking Reveals that ID1 Causes the Formation of Multimers in Wheat Germ Extracts
Figure 17. Limited Crosslinking Reveals that ID1 Causes the Formation of Multimers in E. coli
Figure 18. Batch Purification of Histidine-tagged GAL4 Derivatives
Figure 19. Column Purification of the GAL4 Derivative his-DBD
Figure 20. Column Purification of the GAL4 Derivative his-DBD-AR2
Figure 21. Column Purification of the GAL4 Derivative his-DBD-IDl-AR2
Figure 22. EMSA of Batch Purified Histidine-tagged GAL4 Derivatives
Figure 23. EMSA of Column Purified Histidine-tagged GAL4 Derivatives
Figure 24. Alignment of Protein Sequences Homologous to ID1 of GAL4
Figure 25. Proposed Mechanism of Action of ID1 of GAL4
viii LIST OF ABBREVIATIONS
BSA bovine serum albumin
j
BMV brome mosaic virus
DEPC diethylpyrocarbonate
DMSO dimethylsulfoxide
DSP dithiobis(succinimidylproprionate)
DTT dithiothreitol
ECL enhanced chemiluminescence
EDTA ethylenediaminetetraacetate
EMSA electrophoretic mobility shift assay
Hepes 4-(hydroxyethyl)-l-piperazinethanesulfonic acid
IPTG isopropylthiogalactoside mAb monoclonal antibody
NP-40 NonidetP-40
PAGE polyacrylamide gel electrophoresis
PMSF phenylmethylsulfonyl fluoride
SDS sodium dodecylsulfate
TBP TATA-binding protein ix ACKNOWLEDGEMENTS
I would first like to thank my parents and family for their constant support and encouragement. I am grateful to my husband Steve for always being there for me. I thank Todd Schindeler very much for making the DSP reagent for me, and Logan Donaldson for . the help with the computer homology search and sequence alignment. I also sincerely thank all the members of the Sadowski lab for their help, advice and friendship throughout. I would also like to thank my supervisor, Ivan Sadowski, for his help and guidance. Finally, I would like to acknowledge the help of my committe members including Michel Roberge, Louise Glass, and Ivan Sadowski, for their suggestions on my research and my thesis.
x INTRODUCTION
1. Eukaryotic Transcriptional Regulation
An interesting aspect of molecular biological research focuses on how genes are variably expressed in response to extracellular or developmental signals. These signals generate complex biochemical changes to transduce the signal to the nucleus, and to transcriptionally activate or repress one or more genes. In turn, the change in expression of a gene, primarily at the level of transcription, then allows an appropriate response to the signal.
Transcriptional activators and repressors are mediators between the signal and the response, controlling the level of gene expression. A model of how transcriptional activation occurs is depicted in figure 1. The function of transcriptional activators is to recognize and bind to specific DNA sites called enhancer elements or upstream activating sequences (UAS) found upstream of a gene. Once bound, transcriptional activators interact with proteins of the general transcription initiation complex found at the TATA box, allowing RNA polymerase II to transcribe DNA into RNA (64, 65, 66, 79). Traditionally, it was thought that in all eukaryotes the basal transcription complex was assembled at the promoter in a stepwise fashion (11). Recently, though, it has been shown that in the yeast Saccharomyces cerevisiae, this complex is largely preassembled and termed the RNA polymerase II holoenzyme (42). Koleske and Young (1994) showed that the holoenzyme consists of RNA polymerase II or B, suppressors of RNA polymerase B or SRB's, and the general factors TFIIB, TFIIH, TFIIF. This protein complex then joins TFIIA, TFIID and TFIIE to form the complete general transcription initiation machinery at the promoter. Transcriptional activators function by contacting components of the initiation complex. For example, 34 carboxy-terminal amino acids of the transcription factor GAL4 have been shown to interact with the TATA-binding protein (TBP) and with other coactivators known as SUG1 and ADA2 (58). In addition to the general initiation complex components, other proteins called
1 RNA POLYMERASE II INITIATION COMPLEX
URS TATA BOX
Figure 1. Model of Transcriptional Activation
Transcriptional activation, turning the expression of a gene from off to on, requires several steps. A transcriptional activator binds to its UAS (upstream activating sequence) site on the DNA upstream of a specific gene, changing the conformation of the promoter. The RNA polymerase II general initiation complex is recruited to the TATA box site. The transcriptional activator and the RNA polymerase II general initiation complex interact to allow transcriptional activation of a specific gene.
2 adaptors or coactivators are also found to be essential for transcription, such as the protein GAL11 (31). The mechanism of transcriptional repression is not clear. However, transcriptional repressors have been found to bind DNA at repression sequences or upstream repression sequences (URS) in the promoter of a gene and block transcriptional activation by a largely uncharacterized mechanism (21). Transcriptional activators and repressors have also been shown to play an important role in changing chromatin structure (1, 13, 15), an important aspect in allowing the necessary stimulatory protein-protein interactions to occur (64).
Transcriptional activators are highly modular in nature, allowing researchers the flexibility to swap domains between them while retaining protein function also in other organisms (65, 66, 80). Transcription factors usually contain at least three functional domains to allow for DNA binding, transcriptional activation and regulation activities of the protein. Several structural DNA binding motifs have been identified in both prokaryotic and eukaryotic transcriptional regulators such as zinc finger, leucine zipper, helix- loop-helix and homeodomain motifs (33). Similarly, there are several categories of activating domains such as glutamine rich, proline rich or acidic domains, the latter containing a high proportion of negatively charged residues (65). The regulation of transcription factors usually occurs by either protein-protein interactions or post- translational modification, such as phosphorylation. For example, the S. cerevisiae transcription factor GAL4, which responds to galactose and glucose as extracellular signals, contains a zinc cluster DNA binding motif (57), and two acidic activating domains (56). GAL4 is regulated in part by phosphorylation (71), by the binding of the negative regulator GAL80 (55), and by a glucose-responsive domain which appears to govern the activity of its inhibitory domains (78).
2. Galactose Catabolism in S. cerevisiae
The galactose catabolism pathway of S. cerevisiae is an excellent model for studying the mechanisms of gene regulation.
3 Yeast mutants unable to utilize galactose were first isolated almost forty years ago (69). Since then, the pathway involved and the key players have been defined (17, 18) and continue to be studied intensely. The galactose catabolism pathway, depicted in figure 2, allows yeast to utilize galactose or melibiose, a galactose-glucose disaccharide, as carbon sources, converting them to glucose in order to access glycolysis. The enzymes involved in this conversion are encoded by genes called the GAL genes of the Leloir pathway whose expression, with the exception of GAL5, is controlled by the transcriptional activator GAL4 (18, 40, 41). GAL4 itself is subject to further regulation by the presence or absence of glucose or galactose. In the presence of galactose and the absence of glucose, the preferred carbon source, GAL4 is active and the GAL genes are transcribed at high levels; conversely, in the absence of galactose or in the presence of glucose, GAL4 is inactive and the GAL genes are repressed.
3. Induction by Galactose
As mentioned, GAL4 activity is induced in the presence of galactose but strongly repressed in the presence of glucose. Transcriptional induction of the galactose catabolic pathway genes occurs within minutes of addition of galactose to a yeast culture (17). The actual induction mechanism is not yet fully understood, but it is known to require the interplay of the gene products GAL2, GAL3, GAL1, GAL4 and GAL80. GAL2 acts as the galactose permease, allowing galactose to enter the yeast cell from the medium (59). Once galactose is inside the cell, the GAL3 protein plays an important role in the induction pathway. In fact, yeast strains carrying gal3 mutants are either slow inducers or non-inducers of the galactose catabolism pathway (4, 6). GAL1, which has considerable sequence and functional similarity to GAL3, is capable of replacing GAL3 function (4, 6). GAL3 has been proposed to catalyze the conversion of galactose inside the cell to an inducer molecule, whose target would be the GAL4-GAL80 protein complex. GAL80 binds to GAL4 and acts as a negative regulator to keep GAL4 inactive; thus,
4 ch2oh 4L^L Glu.
>^_^Jr OCH2
MEL1 HO>LHBJ' OH Gal. (out) OH a-galactosidase Melibiose GAL2 \permeas e
HO Gal. (in) GAL1 kinase
HO Gal-1-P HU^I^O- UDP HO GAL7 UDP-Glu. GAL10 transferase epimerase
,o oH0 H UDP-Gal. Glu-1-P u- I GAL5 mutase
HO^L^OH Glycolysis OH Glu-6-P Figure 2. Galactose Catabolism Pathway in S. cerevisiae
The enzymes in this pathway are alpha-galactosidase (MEL1), galactose permease (GAL2), galactokinase (GAL1), galactose-l-phosphate uridyltransferase (GAL7), uridine diphosphoglucose-4-epimerase (GAL10), and phosphoglucomutase (GAL5).
5 perturbation of this protein complex inactivating GAL80 would allow GAL4 to be activated under inducing conditions (4, 6). In fact, gal80~ strains do not require induction. However, since the inducer molecule has not been identified, this model has been modified to consist of a galactose-dependent conversion of GAL3 and GAL1 to forms able of relieving GAL80 negative regulation of GAL4 (5). Without GAL80 inhibition, GAL4 becomes able to activate transcription of the GAL genes.
4. Repression by Glucose
As found in many organisms from prokaryotes to eukaryotes, glucose elicits a stringent repression of several sets of genes in budding yeast (70). One of the sets of genes subject to glucose repression is the GAL gene ensemble (83). This. regulation is energetically advantageous to the cell since glucose can enter glycolysis directly as a carbon source, whereas galactose requires an additional pathway and enzyme activities to first convert it into glucose in order to be utilized as a carbon source. Glucose repression of the GAL genes is a slow process compared with induction, perhaps because the GAL4 protein is quite stable (34). It is also a more complex process, requiring the interplay of many proteins and signals. A summary model of these glucose repression mechanisms is shown in figure 3.
There appear to be four main glucose repression mechanisms (36, 78). The first acts at the level of induction. The expression of both GAL2 and GAL3 is repressed in glucose. The GAL2 permease is also inactivated by glucose through proteolysis (83). Also, the negative regulator GAL80 appears to work better in the presence of glucose, perhaps as a consequence of lack of potential induction by GAL3 (43). This mechanism acts at the binding sites for GAL4 called upstream activating sequences for galactose (UASG) of galactose- inducible genes, and shows that even if galactose is present, glucose is used preferentially.
6 Galactose O Glucose Repression w Mechanism GAL2 H-0
UASG URSG
Figure 3. Mechanisms of Glucose Repression
The glucose repression mechanisms that affect the GAL genes are numbered 1 to 4. Mechanism 1 affects induction, by inhibiting GAL2 and GAL3 and enhacing GAL80 activity. Mechanism 2 inhibits transcription of GAL4 by allowing the MIG1-SSN6-TUP1 repression complex to bind to the URSG sequence found in the GAL4 promoter. Mechanism 3 inhibits transcription of GAL4 regulated genes, like GAL1 shown above, by allowing the MIG1-SSN6-TUP1 repression complex to the URSG sites found upstream of GAL4 regulated genes. Mechanism 4 inhibits GAL4 activity through the central region of GAL4.
7 Glucose also represses GAL gene expression by reducing the level of the GAL4 activator itself. The GAL4 promoter contains GC rich, upstream repression sequences called URSG- The MIG1 protein binds to the URSG (28) and attracts the SSN6-TUP1 repressor complex (82), inhibiting the expression of the GAL4 gene. This inhibition accounts for a powerful and long-term, slow glucose repression effect. Powerful in that GAL4 binds cooperatively to its UASG binding sites (25, 38), and thus a slight change in the amount of GAL4 protein in the cell causes a pronounced effect. Long-term and slow in that the GAL4 protein is quite stable (36), and thus would remain in the cell even without the synthesis of new GAL4 molecules.
A third mechanism by which glucose stringently regulates GAL gene expression is by inhibiting GAL4 function through URSG elements found at the promoters of galactose inducible genes, such as the GAL1 promoter (21). This recruits the binding of the MIG1 protein as is the case for the GAL4 promoter, and, in concert with the SSN6-TUP1 repression complex, these proteins are able to inhibit transcription by an as of yet uncharacterized mechanism. The GAL1 promoter is believed to contain two and possibly three such URSG elements (22). This mechanism is quite rapid, occurring within minutes of glucose addition (36).
The last mechanism of glucose repression acts through the GAL4 protein itself. Stone and Sadowski (1993) showed that the central part of GAL4 contains a glucose-responsive domain termed glucose response domain or GRD that appears to control the activity of three inhibitory domains, also in the central region of GAL4, which prevent GAL4 activity. These observations were made by removing all other known glucose repression mechanisms and still observing 10-fold repression by glucose on GAL4 activity in only twenty minutes. Also, when fused to the chimeric activator LexA-VP16, the GAL4 central region can confer glucose responsiveness to this activator, which is otherwise unresponsive to glucose. Furthermore, if the GRD is removed and only the inhibitory domains of GAL4 are
8 fused to LexA-VP16, inhibition becomes constitutive, regardless of carbon source. This concurs with the idea that glucose controls the GRD, and that, in turn, the GRD controls the activity of the inhibitory domains.
Despite these four main mechanisms, there are several other proteins that play a role in glucose repression of GAL gene expression in an intricate and not yet well defined signalling system. Much of this work has been through genetic findings, but more biochemical work is underway to uncover the details involved. These other proteins include: HXK2, GRR1, REG1, GAL82, GAL83, SNF1, and NGG1. Mutations in HXK2, a hexokinase, were found to result in a loss of glucose repression (54). GRR1 is believed to act on the glucose transporters, acting in a different class of glucose repression genes than REG1, GAL82 and GAL83 (19); REG1 appears to act upstream of GAL82 and GAL83, in an inhibitory way towards these proteins (19). The serine, threonine protein kinase SNF1, in contrast, is required for expression of glucose repressible genes, suggesting a role for phosphorylation in this intricate regulation (14). SNF1 is believed to act downstream of GAL82 and GAL83 and upstream of the MIG1, SSN6, TUPI repressor complex, but its substrates have not yet been defined. It is interesting to note that this repressor complex has been found to contain a DNA binding component, MIG1, which directly interacts with an adaptor, SSN6, which in turn interacts with TUPI, the component believed to carry out the actual repression (84). Also, MIG1 has been found to be phosphorylated when isolated from cells grown in glucose. The MIG1 kinase, though, is still unknown, as is the significance of the phosphorylation of MIG1 in glucose (82). Finally, another protein involved in glucose repression is NGG1, which was shown genetically to be required for glucose repression (9) and through two-hybrid data to interact with parts of GAL4 (8). Once again, though, the exact mechanism involved is still unknown. Glucose repression of the galactose-regulated genes, then, involves a series of mechanisms and players to achieve stringent regulation of gene expression.
9 5. GAL4 Structure and Function
As mentioned, transcriptional activators are highly modular. GAL4 is certainly no exception as can be seen in figure 4. GAL4 was first cloned and analyzed in the early 1980's (37, 44), and subsequently further characterized by revealing its DNA sequence, a 2.8 kilobase polyadenylated RNA transcript, a predicted 881 amino acid encoded protein with a molecular weight of approximately 100 kilodaltons (KDa) (45). Since then, many groups have unveiled a more detailed description of the structure and function of this transcriptional activator. GAL4 is now widely used as a tool in methods such as the two-hybrid system of protein-protein interaction (20), directional binding and selectable expression of fusion proteins, as well as many other techniques (72, 73).
a. DNA Binding
Much work has been carried out to define the DNA binding domain of GAL4. GAL4 was determined to bind to UASG sequences found upstream of galactose-inducible genes. For example, the divergent GAL1-10 promoter bears four such UASG elements, each consisting of a 17 base pair sequence with dyad symmetry and the following conserved motif 5'-CGGNl lCCG-3' (7, 27). As suggested by the dyad symmetry of the 17 mer, GAL4 binds to each site as a dimer (12). The region of DNA binding recognition and specificity was mapped to amino acids 1-65 of GAL4, whereas a dimerization function is provided by amino acids 65-94 (12). Commonly, the first 147 amino acids, which also contain the nuclear localization signal (residues 1-74) (76), are used as the entire GAL4 DNA binding domain, lacking transcriptional activation (39).
The DNA binding motif of GAL4 has been classified as a Zn2Cys6 cluster with six cysteine residues coordinating two zinc ions in order to bind DNA. Recently, the structure of a 65 residue, N-terminal fragment of GAL4 and its binding site has been solved
1 0 100 200 300 400 500 600 700 800 900 1 I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 i ID1 ID2 ID3 GRD GAL4 1m m mm DNA Binding Spedficity —
Dimerizatiori —-:_J- Cooperative DNA Binding h Transcriptional Activation
GAL80 Interaction -
Phosphorylation sites _ i.—
Glucose Response Inhibition Inhibitory Domain 1
Figure 4. Functional Domains of GAL4 and Their Positioning
The locations of the functional domains of GAL4 with respect to the amino acid sequence of the protein are represented by the shaded blocks beside the specified function. The abbreviations used in the linear representation of the GAL4 protein stand for: DNA binding domain (DBD), activating region 1 and 2 (AR1 and AR2), inhibitory domain 1, 2, and 3 (ID1, ID2, and ID3), and glucose response domain (GRD).
1 1 by X-ray crystallography (57). This study reconfirmed that GAL4 binds as a dimer, and showed that the conserved CCG triplet at each end of the DNA site is recognized in the major groove by the Zn2Cys6 cluster. The two fold symmetry is explained by a coiled-coil weak dimerization region perpendicular to the minor groove. The dimerization region is connected by a linker region, which in the case of GAL4 spans 11 base pairs over the minor groove. The findings of this study were confirmed by the results obtained by an NMR solution of the DNA binding domain of GAL4 binding to half of the UASG site as a monomer (2).
The linker region in the GAL4 DNA binding domain is important in the in vivo recognition of the activator's specific sites. In fact, other fungal activators also contain the same zinc requiring DNA binding motif, such as LAC9, the lactose/galactose regulator of Kluveromyces lactis which binds to the same site, PPR1, which regulates the expression of genes involved in pyrimidine biosynthesis in S. cerevisiae and binds to a CCG triplet separated by 6 base pairs instead of 11, and PUT3, which is responsible for controlling genes involved in proline utilization and has a linker region of 10 base pairs (57, 67).
Another important consideration about GAL4 DNA binding is that it does so in a cooperative fashion (25, 38); the exact location of the cooperativity domain of GAL4 is currently under study, but is suggested to be in the C-terminus of the protein (32). This is especially important for a rapid and potent induction response of GAL4, a weakly expressed protein (29, 45).
A final aspect to consider about GAL4 binding to DNA is whether it is always bound at its site, regardless of carbon source available, or whether it is unbound when repressed by glucose. The evidence on this matter is contradictory, with experiments pointing to both results (26, 51). These results may be complicated by the fact that the expression of the GAL4 gene itself is glucose repressed,
1 2 and thus, in the presence of glucose, GAL4 may simply be depleted from the cell.
b. Transcriptional Activation
DNA binding is a necessary but insufficient step for transcriptional activation by GAL4, as was noted by Keegan et al. (1986). In fact, GAL4 contains two acidic domains that are able to activate transcription when fused to the DNA binding domain of the protein; these are termed activating region 1 (AR1, residues 149- 238) and activating region 2 (AR2, residues 768-881) (56). These activation domains were characterized through a series of deletions of GAL4. In addition, there is a third, small activation domain termed activating region 3 (AR3, residues 94-106) that contributes to transcriptional activation in vitro (49). GAL4's activating regions belong to a family of acidic activating domains that many other transcription factors also possess; these all share a high proportion of acidic residues. Mutants of GAL4 have been characterized which indicate that the high acidic content is important in the potency of the activation. (23, 24). These acidic residues also appear to be arranged in an a helical conformation, although some studies contradict these findings by showing that these residues need not be acidic and may not be a helical but may form (3 sheet conformations (47, 85).
Nevertheless, these domains are presumed to contact one or more of the general initiation factors, anchored near the promoter by the DNA binding component of the transcription factor. In fact, it was recently shown that the carboxy-terminal 34 amino acids of GAL4 can interact in vitro with TBP and other general yeast coactivators, such as SUG1 and ADA2 (58). Furthermore, another mechanism by which the activating domains are involved in enhancing transcription is by increasing the ability of basal transcription factors to occupy the promoter region, allowing preinitiation complexes to form after nucleosome assembly (86). Another important aspect of transcriptional activation potency was
1 3 observed recently to be an interplay between additional binding sites for the transcription factor and the additional amounts of transcriptional activation domains in an activator, at least in vitro (61). Finally, as was observed with the positioning of binding sites and respective DNA binding domains of transcription factors in other organisms, the acidic activating regions of GAL4 are also functional in other eukaryotic systems, such as mammalian cells (24). A conserved mechanism of transcriptional activation thus exists throughout eukaryotes, allowing the flexibility to use the activating regions of GAL4 as research tools (72, 73).
c. GAL80 Interaction
It was noticed early on that GAL80 is a negative regulator of GAL gene expression (17, 18), but it was not until later that GAL80 was excluded from being one of the mediators of glucose repression (81). GAL80 was cloned in the early 1980's (37, 44) and was determined to be the negative regulator that acts upon GAL4 itself, a GAL80 null mutation resulting in constitutive GAL expression in the absence of glucose (55).
By deletion experiments, Ma and Ptashne (1987) mapped the region responsible for GAL80 binding to the 30 carboxy-terminal amino acids of the GAL4 protein. Furthermore, it was found that GAL4 and GAL80 coimmunoprecipitate and copurify in an equimolar ratio, further strengthening the notion that these two proteins bind (16, 53, 88). Three main functional domains were found in the GAL80 protein which allow it to function as a negative regulator: a repression domain, an inducer interaction domain, and a nuclear localization domain (60). Interestingly, GAL80 expression is GAL4- induced in the presence of galactose, the GAL80 promoter bearing one UAS.G, but appears to be at a basal level in glucose or glycerol, unlike other GAL4 regulated genes (50). This regulation is probably necessary for the stringent inactivation of GAL4 by GAL80 in non- inducing conditions.
14 Opposing views exist on the matter of whether GAL80 is always bound to GAL4, the GAL4-GAL80 complex being conformationally altered upon induction by galactose, or whether GAL80 simply dissociates from GAL4 under such conditions. Earlier views favoured the dissociation mechanism (55, 63), with GAL80 masking an essential part of GAL4's AR2, but more recent results suggest that the conformational change mechanism is correct in vivo (46, 62).
d. GAL4 Phosphorylation
GAL4 is phosphorylated on several serine residues (71), phosphorylation being a possible regulatory mechanism of protein activity. Unfortunately, the mechanisms involved and the possible kinase or kinases are not yet clearly defined. It is known, however, that, unlike most transcription factors, GAL4 phosphorylation is acquired as a consequence of activity after induction and not as a requirement for transcriptional activation (62, 74). Also, the general transcriptional regulator GAL 11 was observed to augment GAL4 dependent-transcription by maintaining a phosphorylated form of GAL4 (52). An important phosphorylation site appears to be Ser 699, which, when mutated to Ala renders GAL4 a very slow inducer (71). Phosphorylation of Ser 699 appears to prevent GAL80 from repressing GAL4 (71).
e. GAL4 Central Region
Surprisingly, until 1993, no function had been assigned to the large central region of GAL4 (CR, residues 239-767), which comprises over 60% of the entire protein. Through a series of deletion experiments and fusion protein expression and function studies, Stone and Sadowski (1993) were able to show that this region is comprised of at least three inhibitory domains, termed inhibitory domain 1 (ID1, residues 320-412), inhibitory domain 2 (ID2, residues 412-478) and inhibitory domain 3 (ID3, residues 554-585), and of a
1 5 glucose-responsive domain (GRD, residues 600-767), as discussed above.
The conclusions about the GAL4 CR originated from the observations that either AR1 or AR2, when fused to the DNA binding domain, can activate transcription in the absence of the CR (23, 24, 56), but transcription is severely impaired when either AR1 or AR2 is deleted, suggesting an inhibitory, regulatory role for the CR of GAL4 (56, 74, 78). These findings were confirmed by deletion experiments of GAL4 as well as with experiments employing the heterologous transcriptional activator LexA-VP16 (78). When the central region of GAL4 is fused to LexA-VP16, this activator becomes responsive to glucose when it normally is unaffected by the carbon source available. Also, by deleting GRD and fusing the rest of the central region to LexA-VP16, the inhibition becomes constitutive, regardless of carbon source, a result which also seen with the deletion of the GRD from GAL4 itself.
The above results suggest two conclusions. They reconfirm the idea that GAL4 contains a glucose-responsive domain, GRD, which has indeed been shown to be responsible for one of the GAL4 glucose repression mechanisms. They also suggests a model whereby the glucose controlled GRD is in turn responsible for regulating the inhibitory domains, which should be active in glucose and inactive otherwise. The exact mechanism by which this regulation occurs is still unknown, but it was suggested that the GRD and the inhibitory domains interact, whether directly or through other intermediary protein or proteins. This interaction would allow the GRD to keep the inhibitory domains from functioning in the absence of glucose. Upon glucose addition, though, this interaction could be disrupted, the inhibitory domains becoming active to prevent transcriptional activation (77, 78). This model is supported by several missense mutants that have been found between residues 320-520 of GAL4, the inhibitory region, which inactivate the protein (35). This inactivation can be explained by the disruption of GRD-inhibitory domains interaction, rendering inhibition constitutive. Exactly how
1 6 inhibition is achieved is largely unknown, but disruption of DNA binding seems to be a likely mechanism, the strongest evidence presented so far being that LexA derivatives bearing GAL4 CR fusions bind more weakly to DNA when extracted from yeast grown in glucose as compared to other carbon sources (77).
No homology has been found between the three inhibitory domains, and some preliminary results suggest that they have different mechanisms of action (32). Of the three inhibitory domains, ID1 was found to be the strongest and to be quite flexible in terms of its location in a fusion protein while still retaining activity (77). ID1 is also the site of three point mutants at amino acids 322, 331 and 352 which, along with several DNA binding mutants and one at residue 511, can severely hinder GAL4 activity (35). These mutants suggest that ID1 is an important regulatory region. This domain, residues 320-412, also contains a high degree of homology with other fungal transcription factors involved in the regulation of metabolic pathways such as LAC9, PUT3, PPR1, LEU3, NIRA, THI1 and NIT4, as well as other proteins such as CDC6, involved in initiation of DNA replication, and PDR3, involved in pleiotropic drug resistance. The significance of this homology is not yet understood, but it suggests the interesting possibility of a conserved functional mechanism by which inhibition can be achieved.
6. Aim of Research
The aim of my research was to determine the mechanism of action of inhibitory domain 1 (ID1) of GAL4. With biochemical evidence, I suggest that the transcriptional inhibition activity posessed by ID1 occurs through a protein multimerization mechanism. This protein multimerization prevents efficient formation of GAL4 dimers, and thus impairs the DNA binding ability necessary for the activity of the GAL4 transcriptional activator.
1 7 MATERIALS AND METHODS
1. Ribonuclease Protection Assay
RNA Extractions: Yeast strain YT6::171 derived from YT6 (MATa, gal4, gal80, ura3, his3, ade2, adel, lys2, trpl, aral, leu2, met)
(31) with a wild-type GALl-lacZ reporter gene bearing both UASG and URSG (87) was used for RNA extractions. YT6::171 was transformed by the lithium acetate method with ARS-CEN plasmids yCPG4trp, yCPG4242 and ycplac22 (constructed by I. Sadowski) expressing wild type GAL4, GAL4 without the central region and a vector control, respectively, from GAL4's own promoter. 20 ml yeast cultures were grown from these transformants in minimal medium lacking tryptophan and uracil and having glycerol, lactic acid and ethanol as carbon sources. At OD600=l-5, 2% glucose was added to half of the cultures, and all cultures were grown for another hour. 10 ml of each culture (either with or without glucose) was for RNA extraction. RNA was extracted by pelleting the cells, washing with 1 ml sterile, deionized water, resuspending in 500 uL Ultraspec RNA (Biotecx) and adding 400 uL glass beads before vortexing 40 minutes at 4° C. 100 jiL chloroform was then added. The samples were incubated for 5 minutes on ice, centrifuged 5 minutes at 4°C, and the top aqueous layer removed for isopropanol freezer precipitation overnight. The pellet was then recovered by centrifugation, washed with 70% ethanol, and resuspended in water treated with diethylpyrocarbonate (DEPC). A sample was taken to determine the concentration by A260 reading.
Ribonuclease Protection Assay: The lacZ and actin probes used in this assay were derived from linearized plasmids pIS009 and pIS015 (constructed by I. Sadowski). Each template was transcribed in vitro with SP6 RNA polymerase and gel purified as described in the manual for the RPAII kit sold by Ambion. The rest of the assay was also performed as described in the RPAII kit manual, using 1 pg RNA per reaction.
1 8 2. (3-Galactosidase Assays
P-galactosidase assays were performed on strain YT6::171. This strain was transformed with high copy 2|i plasmids pMA210, pMA241, pMA236, pMA200 (contructed by J. Ma), and pMAIDl (derived from pMA210, substituting the Xho I-Mlu I fragment with that of plasmid pkwlO(Bam)A312:A421 constructed by G. Stone). These plasmids express wild type GAL4, GAL4 1-147 DNA binding domain (DBD), GAL4 DBD fused to AR2, a vector control, and GAL4 DBD fused to ID1 fused to AR2, respectively, from an ADH1 promoter. The transformants were grown in histidine-, uracil" dropout medium. The assays were performed as described by Himmelfarb et al. (1990), the results expressed as Miller units (1 unit= 1 pmol o- nitrophenol consumed/ min/ mg protein).
3. DNA Binding Assays
Expression in E. coli: BL21 E. coli cells which express T7 RNA polymerase were transformed with plasmids pTMC147, pTMCld768, pTMCl-147-IDl-II, pTMCld683, and pRB451 (contructed by I. Sadowski, I. Sadowski, I. Sadowski, T. Kang, and R. Brent, respectively) expressing GAL4's DBD, DBD fused to AR2, DBD fused to ID1 fused to AR2, DBD fused to GRD fused to AR2, and a vector control, respectively, from a tac promoter. 50 ml of LB broth medium with 1 mg/mL ampicillin was inoculated with overnight cultures of the individual transformants, and grown until OD550 was 0.7. The cultures were then induced with 1 mM isopropylthiogalactoside (IPTG) for 3 more hours. Crude extracts were subsequently made by spinning down the cells, washing them with cold E. coli extraction buffer (20 mM 4-(hydroxyethyl)-l- piperazinethanesulfonic acid or Hepes at pH 7.5, 0.2 M NaCl, 10% glycerol, 0.1 mM dithiothreitol or DTT, 1 mM phenylmethylsulfonyl fluoride or PMSF), resuspending the cells in 2 mL of extraction buffer, sonicating for a total time of 3 minutes, centrifuging the sonicates for 20 minutes at 12100xg, aliquotting and flash freezing
19 the supernatants. The crude extracts were stored at -70°C until time of use.
Cloning for Expression In Vitro: Plasmids pG147+AR2 and pGIDl for expression in vitro of GAL4's DBD fused to AR2 and DBD fused to ID1 fused to AR2 were subcloned from the pGEM3(-) derivative pGWT (contructed by I. Sadowski). pG147+AR2 was made by replacing the Xho I-Mlu I fragment of pGWT with that of pMA236 (constructed by J. Ma); the Xho I-Mlu I of pkwlO(Bam)A312:A421 (constructed by G. Stone) replaced that of pGWT to make pGIDl. PCR reactions were set up using 0.01 u\g of each Nde I linearized plasmid (pG147+AR2 and pGIDl) to make DNA templates of defined sizes to be used for in vitro transcription reactions. Vent DNA Polymerase was used in standard 100 pL PCR reactions using 55 pmol of each primer CP2 (5' -CACAGTTGAAGTGAACTTGC-3') and SP6P/P (5'- CATACGATTTAGGTGACACTATAG-3'). The PCR products were purified using Promega's Wizard^M PCR* kit and resuspended in 100 pL DEPC-treated water. Plasmid pG241 (constructed by I. Sadowski), derived from pGEM3(-) and expressing GAL4's DBD, was also used as a template for in vitro transcription reactions by linearizing it with Ndel.
In Vitro Transcription Reactions: The templates described above were used to synthesize GAL4 derivative RNA in vitro. The 50 pL reactions were performed as described in Promega's "Transcription In Vitro" technical publication and contained 10 pL of the PCR products described above or 2 jxg of Nde I linearized pG241 as templates and SP6 RNA polymerase as enzyme.
In Vitro Translation Reactions and Protein Normalization: The reactions were carried out in the wheat germ extract system sold by Promega by adding 5 pL of the in vitro translated products described above to a standard translation reaction with 6 pCi [35S]-methionine label as described in Promega's "Translation In Vitro" technical publication. For co-translation reactions, 5 u.L of each two chosen types of RNA were added instead of a single type in normal
20 translation reactions. To normalize the amount of protein of interest subsequently used in further experiments, 2 jxL samples of each completed reaction were run on a 12% SDS-PAGE gel. The gel was then Coomassie blue stained, fixed, enhanced with EN^HANCE (Dupont), swollen with water and 10% glycerol, dried and exposed to X-ray film overnight. The bands of interest were cut out of the gel and counted by liquid scintillation. The number of counts obtained was then normalized to mmol of protein per methionine residue by using the specific activity of the [35s]-methionine label (1175 Ci/mmol), from which an equimolar ratio of protein was calculated, knowing that 1 Ci=2.2xl012 dpm.
Electrophoretic Mobility Shift Assays: These assays were done using either crude E. coli extracts, purified E. coli produced proteins (described below) or wheat germ extracts, all expressing specific proteins. Each reaction contained equal amounts of specific protein as previously determined by Western blotting, SDS-PAGE Coomassie Blue staining or liquid scintillation counting. Binding reactions were performed in Ficoll buffer (20 mM Hepes pH 7.5, 5 mM MgCl2, 100 fig/mL bovine serum albumin or BSA, 2.5% ficoll, 10 u,M ZnSo4, 50 mM NaCl, 0.1 mM DTT, 0.1 mM ethylenediaminetetraacetate or EDTA)
for 30 minutes on ice with 4 pmol of [32p]_iabelled double stranded GS3/GS4 GAL4 oligo (5'-TCGACGGAGGACAGTACTCCG-3') prior to analysis on 4.5% non-denaturing gels as described by Carey et al. (1989).
GAL4 Biotinylated Oligo Binding Assay: These assays were done using proteins expressed either in E. coli as crude extracts or in vitro in wheat germ extracts. For in vitro produced proteins, the experiment was carried out as follows: equimolar amounts of protein (as predetermined by liquid scintillation counting) were incubated with 25 u\L of pre-washed streptavidin agarose beads (Gibco-BRL) at 4°C on a rocker for 20 minutes as a preclearing step. The supernatants were then incubated with 100 pmol of GAL4 biotinylated oligo (5' -biotin-CGGAGTACTGTCCTCCG-3'), 150 pL of buffer 2xBIO (50 mM Hepes pH 7.5, 10 mM MgCl2, 300 mM KC1, 0.2
2 1 mM EDTA, 20% glycerol, 0.2 mM DTT, 2 mM PMSF, 20 pg/mL aprotinin, 2 pM leupeptin), 25 pg casein, and 20 pg poly-dldC in a total 300 pL reaction for 30 minutes on a rocker at room temperature. 100 pL of washed streptavidin agarose beads was then added, and the mixture further incubated for 30 minutes on a rocker at 4°C. The incubated beads were then washed four times with 1 mL of buffer lxBIO, transferring tubes on the last wash. The beads were then resuspended in 10 pL of 2xSDS sample buffer, boiled for 5 minutes and centrifuged for 5 minutes. The supernatant was then loaded on a 12% SDS-polyacrylamide gel which was then subjected to electrophoresis, Coomassie blue staining, fixing, enhancing with EN^HANCE, and swelling with water and 10% glycerol, before being dried and exposed to X-ray film. For E. coli produced proteins, the assay was performed similarly except that equal amounts of protein (as predetermined by Western blotting) were added directly to the 300 pL reaction (without a preclearing step) which lacked poly-dldC and whose 2xBIO buffer contained 50 mM Hepes pH7.5, 10 mM MgCl2, 100 mM KC1, 0.2 mM EDTA, 20% glycerol, 0.2 mM DTT, 2 mM PMSF, 20 p g/mL aprotinin, and 2 pM leupeptin. The remaining incubations were as described above as were the four washes in lxBIO buffer for E. coli. There was no tube transfer at the end of wash four. The supernatant derived from the resuspension, boiling and centrifuging of the beads in 2xSDS sample buffer was also loaded on a 12% gel which was subjected to electrophoresis and then transferred onto nitrocellulose for Western blotting with mAb 5c8- 12 to GAL4's DBD, and ECL detection as described by Ambion.
4. Chemical Crosslinking Experiments
The thiol cleavable, homobifunctional chemical crosslinker dithiobis(succinimidylpropionate) or DSP (Pierce chemicals) was used in all chemical crosslinking reactions. The stock reagant was prepared in dry dimethylsulfoxide (DMSO) by Todd Schindeler (U.B.C. Chemistry Department) to a concentration of 25 mM and stored at 4°C. Equimolar amounts of protein of interest from either E. coli or wheat germ extracts were added to a 100 pi total volume reaction
22 consisting of DSP reaction buffer (20 mM sodium phosphate pH 7.5, 0.15 M NaCl) and, unless otherwise stated, 1 mM DSP crosslinker. The reaction was allowed to proceed at room temperature for 30 minutes and then quenched with 50 mM Tris, pH 8.0 for a further 30 minutes. For crosslinking with E. coli proteins, the reactions were then divided in half; 2xSDS sample buffer containing (3-mercaptoethanol was added to one half of the sample, and 2XSDS sample buffer lacking P-mercaptoethanol was added to the other half. All reactions were then boiled and subjected to SDS-PAGE, transferred onto nitrocellulose and Western blotted with GAL4 mAb 5c8-12 to GAL4's DNA binding region. For in vitro synthesized proteins, the crosslinked reactions were diluted to 500 JIL in lxRIPA buffer (100 mM Tris pH8.0, 100 mM NaCl, 1 mM EDTA, 1% Nonidet- P-40, 0.5% sodium deoxycholate, 0.1% SDS, ImM PMSF, 10 ug/ml aprotinin, 1 pM leupeptin) following crosslinking, and then immunoprecipitated. The immunoprecipitations were performed by first preclearing with 50 uX 10% formalin fixed Staphylococcus aureus (S.A.) for 30 minutes at 4°C, then incubating the diluted reactions with 20 pL mAb 5c8-12 for one hour on ice, adding 50 pL rabbit-anti-mouse (RAM) coated 10% S.A. and incubating for one hour on a rocker at 4°C. After centrifugation, the pellet was washed four times with 1 mL lxRIPA buffer, the pellet being split into two equal portions upon the last wash. One half of the pellet was then resuspended in 2xSDS sample buffer containing P-mercaptoethanol and the other half in 2xSDS sample buffer lacking P-mercaptoethanol. The suspensions were incubated 10 minutes at 37°C, centrifuged for 5 minutes and the supernatants subjected to SDS-PAGE. The gels were then Coomassie blue stained, fixed, enhanced (EN^HANCE) and swollen with water and 10% glycerol before being dried and exposed to X-ray film.
5. Experiments with E. coli Histidine-tagged GAL4 Derivatives
Cloning and Expression: The histidine-tagged GAL4 derivative expression plasmids were subcloned from the pBIDOMAIN plasmid (expressing GAL4's DBD fused to AR2 and tagged with six histidine
23 residues) constructed by W. Crosby in pRSET B (Invitrogen). The pBIDOMAIN plasmid was digested with EcoR I and Hind III, treated with Klenow and religated to give pHISDBD, expressing a histidine- tagged version of GAL4's DBD. To obtain pHISIDl, a histidine-tagged version of GAL4's DBD fused to ID1 fused to AR2, the pBIDOMAIN Xho I-Mlu I fragment was replaced with that of pkwlO(Bam)A321:A412 (constructed by G. Stone). The clones were transformed into NM522 E. coli cells ([proAB+lacIqZAM15] supE thi-1 A(lacproAB)' Ahsd (rmj Ar deoR+) (donated by D. Kilburn) for expression. Expression of the histidine-tagged fusion proteins was achieved as follows: 250 mL of fresh LB medium with 1 mg/mL ampicillin was inoculated with a 1/100 dilution of an overnight culture. The cells were grown to OD600 of 0.3, and then ImM IPTG was added. At OD600=0.6, M13T7-RNAPol phage expressing T7 RNA polymerase (obtained from W. Crosby) was added to a multiplicity of infection of 5 per cell. The cultures were further induced for 4 hours. The cells were harvested by centrifugation. The pellet was washed with XA90 lysis buffer (10 mM Tris pH 8.0, o.5 M NaCl, 10% glycerol, 10 mM P-mercaptoethanol, and 0.1% Tween-20), and then resuspended in 4 mL of the same buffer before sonicating for 4 minutes. The supernatant was cleared by centrifugation (15 minutes, 27200xg), aliquoted, flash frozen and stored at -70°C until time of use. Samples were routinely screened by Western blotting for protein expression levels.
Batch Purification: Crude E. coli extracts expressing GAL4 histidine-tagged derivatives were purified in a batch procedure as follows. The extracts were incubated with 500 pL of ProBond Ni^+- NTA agarose beads (Invitrogen) equilibrated in buffer A (20 mM Tris pH 8.0, 100 mM KC1, 20% glycerol, 30 mM imidazole) for 30 minutes on a rocker at 4°C. The beads were then washed 4 times with 5 mL cold buffer A. The proteins were then eluted with 400 pL of buffer B (20 mM Tris pH 8.0, 100 mM KC1, 20% glycerol, 250 mM imidazole). Samples of the purified proteins were then analyzed by SDS-PAGE and Western blotting. This procedure was adapted from Reece, Rickles and Ptashne (1992).
24 Column Purification: A 2 mL ProBond resin column equilibrated in buffer A (composition described above) was used to purify the histidine-tagged GAL4 derivatives from crude E. coli extracts (68). An Econo Sytem purification apparatus (Bio-Rad) was used at 4°C, with a flow rate of 0.5 ml/min. 1 mL fractions were collected while monitoring A280- The elution gradient ranged from buffer A described above at 30 mM imidazole to buffer B described above at 500 mM imidazole. The duration of the elution gradient ranged from 1 hour for pHISDBD extracts, to 20 minutes for pBIDOMAIN extracts, to 40 minutes for pHISIDl extracts. Samples of the column fractions were analyzed by SDS-PAGE and Coomassie blue staining.
25 RESULTS
1. Glucose Inhibits GAL4 Through Its Central Region
As described by Stone and Sadowski (1993), the central region of GAL4 contains a glucose-responsive domain called GRD and three inhibitory domains (ID1, 2, 3). To further characterize the effect of glucose on the GAL4 protein, I performed a ribonuclease protection assay. The analysis of mRNA production instead of protein activity in response to glucose was necessary since G. Stone showed with nuclear run-on transcription assays that glucose inhibition of GAL4 through its central region has rapid kinetics, peaking within 20 minutes, the GAL4 protein recovering with time (77).
Yeast RNA was extracted from cultures grown in glycerol with or without the addition of 2% glucose for one hour prior to extraction. The yeast strain used was YT6::171 which contains UASG and URSG sites upstream of a GAL1 -lacZ reporter gene. The strain expressed either wild type GAL4 protein (plasmid yCPG4trp), GAL4 protein lacking the central region (CR, residues 320-767; plasmid yCPG4242) or a vector control (ycplac22) from GAL4\ wild type promoter. 1 pg of each RNA extracted, as measured by optical density, was hybridized to lacZ and actin probes. These probes were used to detect transcriptional activation of the reporter gene as well as production of an internal control to quantitate more accurately the amount of RNA in each sample.
The results of the ribonuclease protection assay are depicted in Figure 5. As can be seen, both wild type GAL4 and GAL4 CR deletion could activate transcription in yeast growing in glycerol. But, when yeast were subjected to 2% glucose for one hour, wild type GAL4 became completely inactive whereas GAL4 CR deletion was not affected by the presence of glucose, and, actually, even showed about 10 fold induction. The vector control behaved as expected, showing no transcriptional activation of the lacZ reporter gene in either glycerol or glucose.
26 glycerol glucose lacZ m f f
actin - m *» • 1 2 3 4 5 6 7 8
Figure 5. Glucose Represses GAL4 Through Its Central Region
Ribonuclease protection assay performed with 1 pg total yeast RNA probed with lacZ and actin probes. The yeast RNA was extracted from strain YT6::171 expressing, from a wild type GAL4 promoter, either wild type GAL4 (lanes 3 and 6), GAL4 lacking its central region (lanes 4 and 7) or a vector control (lanes 5 and 8). The yeast cultures were grown in glycerol until OD600=l-5, then separated into two, one half receiving 2% glucose for one hour before extraction (lanes 6-8), the other half receiving no glucose (lanes 3-5). Lanes 1 and 2 were positive yeast RNA controls probed with actin or lacZ probes alone, respectively. This experiment, as well as all others, were repeated with reproducible results at least twice, except where indicated.
27 This ribonuclease protection assay result, then, reconfirmed that the GAL4 protein is indeed rapidly affected by glucose through its central region as described by Stone and Sadowski (1993). Also, this result establishes glucose inhibition of the GAL4 protein as an important and relevant glucose repression mechanism, unlike what Johnston et al. (1994) stated. This glucose repression mechanism remains to be further characterized by determining how the signal from glucose is relayed to the central region of GAL4 and how the GRD controls the activity of the inhibitory domains as has been proposed (78).
2. ID1 Is a Strong Inhibitory Domain in Yeast
The next step of my research involved reconfirming that GAL4's ID1 (residues 320-412) is a strong inhibitory domain in vivo in yeast cells, as observed by Stone and Sadowski (1993). Again, I used the yeast strain YT6::171 with a GALl-lacZ reporter gene to assay transcriptional activation by different GAL4 derivatives in the form of p-galactosidase activity in dropout, selective medium. The GAL4 derivatives, depicted in figure 6, consisted of wild type GAL4, GAL4 DNA binding domain (DBD, residues 1-147), GAL4 DBD fused to AR2 (DBD-AR2), GAL4 DBD fused to ID1 fused to AR2 (DBD-ID1-AR2), as well as a vector control (plasmids pMA210, pMA241, pMA236, pMAIDl, and pMA200, respectively). All of these derivatives were overexpressed from an ADH1 promoter contained in high copy 2 p plasmids.
The results of the P-galactosidase assays are shown in table 1. As can be seen, the wild type GAL4 and no GAL4 controls showed the expected high activity and no activity, respectively. Also as expected, DBD was inactive as it can only bind DNA, lacking transcriptional activation activity which was seen in the DBD-AR2 construct. On the other hand, transcriptional activation activity was lost with the DBD-ID1-AR2 construct.
28 1 100 200 300 400 500 600 700 800 900 wild type GAL4 ID1 ID2ID3 GRD mm rfY/y/yy/y/A
DBD
DBD-AR2 DBD -M
DBD-ID1-AR2 ID1
Figure 6. GAL4 Derivatives
The makeup of three GAL4 derivatives called DBD (DNA binding domain), DBD-AR2 (DNA binding domain fused to activating region 2) and DBD-ID1-AR2 (DNA binding domain fused to inhibitory domain 1 fused to activating region 2) is depicted in a linear form with respect to wild type GAL4. The scale at the top numbers the amino acid residues in the proteins. The dashed line (—) joining the different parts of the GAL4 derivatives represents a direct fusion between these parts. These derivatives were expressed in yeast, E. coli and wheat germ extracts.
29 GAL4 Derivative p-Galactosidase Activity (Miller units ± SD)
Wild Type GAL4 1505 ±168
No GAL4 <1
DBD <1
DBD-AR2 1373 ±43
DBD-ID1-AR2 <1
Table 1. p-Galactosidase Assay of GAL4 Derivatives
The (3-galactosidase activities were assayed in the yeast strain YT6::171 transformed with plasmids over-expressing the GAL4 derivatives listed above. The results are expressed as Miller units plus or minus the standard deviation (SD) of the duplicate samples.
30 The (3-galactosidase assays in table 1 show that ID1 is responsible for strong in vivo inhibition of GAL4 in yeast, as was observed by Stone and Sadowski (1993). Stone and Sadowski suggested that GAL4's DNA binding ability was affected by the inhibitory action of the CR but had not elucidated a clear mechanism of action of the inhibitory domains. They also noted that ID1 was the strongest inhibitory domain, and that it contained homology to other fungal transcription factors. For these reasons, I set out to characterize the mechanism of action of ID1 in my research plans.
3. ID1 Impairs DNA Binding
The first question to be answered was whether ID1 affects GAL4 DNA binding as was suggested (78), or whether it impairs GAL4 functioning while GAL4 is bound to DNA. DNA binding of a transcriptional activator is a necessary but insufficient step to achieve transcriptional activation. I used two approaches to resolve this DNA binding question: electrophoretic mobility shift assays (EMSA) and GAL4 biotinylated oligo assays.
I performed EMSA experiments with GAL4 derivatives expressed in E. coli as crude extracts, seen in figure 7, and with GAL4 derivatives expressed in vitro in wheat germ extracts, seen in figure 8. The amounts of each protein used were standardized to comparable amounts by Western blotting for E. coli expression, and to exact amounts by liquid scintillation counting for in vitro expression. Even though Western blotting was performed with a GAL4 monoclonal antibody, some background bands were consistently observed, even with the no GAL4 control extract (figure 7, lane 4), suggesting that these bands result from some cross- reactivity of the antibody with components of the E. coli crude extract. These background bands were observed particularly when the amount of the crude extract tested was increased to normalize the protein of interest, such as lanes 3, 4, and 5 in figure 7, as opposed to lanes 1 and 2, which contained less crude extract.
3 1 Figure 7. Expression of GAL4 Derivatives in E. coli
Western blot of crude E. coli extracts separated on a 12% SDS-PAGE gel, probed with mAb 5C8-12 to the GAL4 DNA binding domain, and detected by ECL. The GAL4 derivatives expressed in the extracts are DBD (lane 1, position a), DBD-AR2 (lane 2, position b), DBD-ID1-AR2 (lane 3, position c), no GAL4 vector control (lane 4), and DBD-GRD- AR2 (lane 5, position c).
3 2 Figure 8. Expression of GAL4 Derivatives in Wheat Germ Extracts
Autoradiogram of a 12% SDS-PAGE gel showing [35s]-methionine labelled GAL4 derivatives translated in vitro in wheat germ extracts. The GAL4 derivatives are: DBD (lane 1, position a), DBD-AR2 (lane 2, position b), and DBD-ID1-AR2 (lane 3, position c). Luciferase protein (lane 4, position d) was also expressed in wheat germ extracts as a control.
33 As can be seen in figure 9, the E. coli GAL4 derivative consisting of DBD showed the expected strong binding ability to its GAL4 labelled binding site. Similarly, DBD-AR2, a transcriptionally active GAL4 derivative as shown above by (3-galactosidase assay, also bound DNA. But, with the DBD-ID1-AR2 fusion protein, DNA binding ability was severely impaired when compared to all other derivatives. In this case, no DNA binding was observed even with the use of three times the amount of protein compared to the other derivatives, as can be seen by the increasing breakdown product, also present with the other GAL4 derivatives (Figure 9, position a). I speculate that this breakdown product, seen consistently in all EMSA experiments done with E. coli extracts, consists of a minimal DNA binding GAL4 derivative, thus the band shift. A transcriptionally active control GAL4 derivative with a similar molecular weight to DBD-ID1-AR2 (figure 7), consisting of GAL4's DBD fused to GRD, instead of ID1, fused to AR2 (DBD-GRD-AR2) (77), did not show altered DNA binding. This result suggests that it was not the addition of any GAL4 sequence placed between DBD and AR2 that caused the DNA binding impairement, but a direct result of the presence of ID1. Other controls were also tested. A baculovirus produced wild type GAL4 extract (made by T. Kang) bound DNA as expected, whereas an E. coli extract expressing no GAL4 did not bind to the oligo, showing specificity of the observed DNA binding.
EMSA results with GAL4 derivatives produced in vitro in wheat germ extracts shown in figure 10 support the EMSA results obtained with E. coli produced GAL4 derivatives shown in figure 9. Equimolar amounts of the different derivatives were used in the binding reactions. As seen with E. coli GAL4 fusions, in vitro synthesized DBD and DBD-AR2 were able to bind to the GAL4 specific oligo, whereas DBD-ID1-AR2 was not. DNA binding of the ID1 containing derivative was impaired at equimolar amounts of protein compared to the other two derivatives as well as with twice the amount of DBD-ID1-AR2 protein. A wheat germ extract containing no RNA upon translation as well as one containing Brome Mosaic Virus (BMV) RNA (provided by Promega), which translates into five proteins, showed no DNA
34 Figure 9. ID1 Impairs DNA Binding in E. coli in EMS A
EMSA reactions were performed with equal amounts of crude E. coli extracts containing GAL4 derivatives and a GAL4-specific oligo. The GAL4 oligo alone is shown in lane 1. The GAL4 derivatives added to the binding reactions are: DBD (lane 2, position b), DBD-AR2 (lane 3, position c), DBD-ID1-AR2 at IX, 2X and 3X concentrations (lanes 4, 5, 6, respectively), no GAL4 vector control (lane 7), and DBD-GRD-AR2 (lane 9, position d). Wild type GAL4 expressed from baculovirus was also used as a positive control (lane 8, position e). Position a points out the protein breakdown product forming a GAL4 minimal DNA binding domain seen in all reactions containing GAL4 derivatives.
3 5 36 Figure 10. ID1 Impairs DNA Binding of In Vitro Translated GAL4 in EMSA
EMS A reactions were performed with equimolar amounts of [^S]- methionine labelled GAL4 derivatives expressed in wheat germ extracts to a GAL4 specific oligo. The GAL4 oligo alone is shown in lane 1. The GAL4 derivatives added to the binding reactions were: DBD (lane 2, position a), DBD-AR2 (lane 3, position b), and DBD-ID1- AR2 at IX and 2X concentrations (lanes 4 and 5). Lanes 6 and 7 were negative controls of wheat germ extract expressing no protein or BMV proteins, respectively.
3 7 1 2 3456 7
38 binding, suggesting that the DNA binding seen with DBD and DBD- AR2 derivatives was specific.
To further confirm the DNA binding results suggested by the EMSA data, I performed experiments with a biotinylated GAL4 UASG oligo. Any GAL4 derivative bound to this oligo was isolated with the use of streptavidin agarose beads, whereas all unbound material was washed away. Once again, I tested both E. coli and in vitro expressed GAL4 fusion proteins with this DNA binding assay.
As shown in figure 11, the E. coli made DBD, DBD-AR2 and DBD- GRD-AR2 GAL4 derivatives (expression shown in figure 7) bound to the GAL4 biotinylated oligo. On the other hand, the DBD-ID1-AR2 derivative, whose molecular weight is very similar to the DBD-GRD- AR2 derivative, was not retained by the beads, implying that this GAL4 fusion had not bound to the GAL4 oligo. The DNA binding that was observed, for example with DBD, was specific to the GAL4 oligo, in that if the GAL4 biotinylated oligo was omitted from the reaction, no DBD protein was retained. Once again, the presence of ID1 appeared to impair the protein's ability to bind DNA.
Similarly, GAL4 biotinylated oligo assay performed with GAL4 fusions translated in vitro in wheat germ extracts, displayed in figure 12, also confirmed the results obtained with E. coli expressed proteins (figure 11). Both the DBD and the DBD-AR2 derivatives were specifically retained on the oligo bound to the beads, but the DBD-ID1-AR2 derivative was not. If the oligo was omitted from the reactions, no derivative bound, as expected. As a control for specificity, luciferase (RNA supplied by Promega) in vitro translated protein did not bind to the GAL4 oligo, also as expected.
The results obtained with EMSA and GAL4 biotinylated oligo assays with both E. coli and wheat germ in vitro expressed proteins, then, suggest that the presence of ID1 severely impairs DNA binding of GAL4 derivatives, probably to the order of 100-fold. This result suggests that ID1 inhibits GAL4 from binding to DNA instead of
39 oligo + + - + + + 55^™ • -r C 36— 26— b
1 2 3 4 5 6 7
Figure 11. ID1 Prevents DNA Binding in E. coli to a GAL4 Biotinylated Oligo
Western blot of a DNA binding assay performed with GAL4 derivatives expressed in E. coli to a GAL4 specific biotinylated oligo isolated with streptavidin agarose beads. All unbound material was washed away with four washes of 1XBIO buffer. The GAL4 derivatives used in this DNA binding assay are: DBD (lanes 1 and 2, position a), DBD-AR2 (lane 3, position b), DBD-ID1-AR2 (lanes 4 and 5), no GAL4 vector control (lane 6) and DBD-GRD-AR2 (lane 7, position c). The biotinylated oligo was omitted from the reactions in lanes 1 and 4 as a control for binding specificity.
40 oligo + - + - + - + - 63 _ 53 — 36 — 32 —
1 2 3 4 5 6 7 8
Figure 12. ID1 Prevents DNA Binding in Wheat Germ Extracts to a GAL4 Biotinylated Oligo
Autoradiogram of a DNA binding assay performed with [^^S]- methionine labelled, in vitro translated GAL4 derivatives to a GAL4 specific biotinylated oligo isolated with streptavidin agarose beads. All unbound material was washed away with four washes of 1XBIO buffer. The GAL4 derivatives used in this DNA binding assay are: DBD (lanes 1 and 2, position a), DBD-AR2 (lanes 3 and 4, position b) and DBD-ID1-AR2 (lanes 5 and 6). Luciferase protein translated in vitro in wheat germ extract was also used as a negative control (lanes 7 and 8). The GAL4 biotinylated oligo was either added to or omitted from the binding reactions as indicated as a control for binding specificity.
4 1 inhibiting GAL4 while bound to DNA. The next step to be resolved was how ID1 was affecting DNA binding.
4. ID1 Prevents Dimerization
A possibility of how ID1 may impair DNA binding is by preventing dimerization of GAL4 molecules. I set out to examine this possibility by chemical crosslinking experiments using in vitro translated GAL4 derivatives DBD, DBD-AR2 and DBD-ID1-AR2 from wheat germ extracts. The crosslinker used for these experiments was the thiol cleavable, homobifunctional DSP reagent. With this reagent, I examined both homodimer and heterodimer formation of GAL4 derivatives.
For homodimer analysis, equimolar amounts of each in vitro synthesized GAL4 fusion (a typical in vitro synthesis is shown in figure 8) were added to the crosslinking reaction. The GAL4 species were then isolated by immunoprecipitation and analyzed by SDS- PAGE, either in a crosslinked state using a sample loading buffer lacking P-mercaptoethanol or in an uncrosslinked state using a sample loading buffer containing P-mercaptoethanol. The results of this experiment are depicted in figure 13. Figure 13 shows that both DBD and DBD-AR2 formed homodimers when crosslinked, whereas DBD-ID1-AR2 was unable to form homodimers. Instead, DBD-ID1- AR2 appeared to form large protein complexes that were retained at the top of the gel. Since GAL4 binds as a dimer to its 17 mer dyad symmetrical site (12), lack of homodimer formation caused by the presence of ID1 would necessarily impair DNA binding, a result concurring with my previous observation about the mechanism of action of ID1.
I also tested whether ID1 would prevent the formation of heterodimers. First, all paired combinations of the three GAL4 derivatives DBD, DBD-AR2, DBD-ID1-AR2 were co-translated in vitro in wheat germ extracts, as shown in figure 14. Subsequently, these co-translated proteins were subjected to DSP crosslinking,
42 Figure 13. ID1 Prevents the Formation of Homodimers
Autoradiogram of a crosslinking experiment examining the formation of homodimers of [35s]-methionine labelled GAL4 derivatives expressed in wheat germ extracts. Equimolar amounts of GAL4 derivatives were crosslinked with DSP, immunoprecipitated with mAb 5C8-12 to the DNA binding domain of GAL4 and separated by 10% SDS-PAGE either in the crosslinked state without the addition of P-mercaptoethanol (P-Me) to the sample loading buffer (lanes 1-3), or in the uncrosslinked state with the addition of P-mercaptoethanol to the sample loading buffer (lanes 4-6). The GAL4 derivatives used were: DBD (lanes 1 and 4), DBD-AR2 (lanes 2 and 5), and DBD-ID1- AR2 (lanes 3 and 6). The species indicated by the arrows represent: DBD monomers and dimers (positions a and b, respectively), DBD-AR2 monomers and dimers (positions c and e, respectively), and DBD-ID1- AR2 momoners (position d).
4 3 p-Me + + + 200—1 116—' 97— 66—
45
29
1 2 3 4 5 6
44 53 36; c 32 b
1 2 3
Figure 14. In Vitro Co-translation of GAL4 Derivatives in Wheat Germ Extracts
Autoradiogram of a 12% SDS-PAGE gel showing the [35s]-methionine labelled, co-translated GAL4 derivatives DBD (position a), DBD-AR2 (position b) and DBD-ID1-AR2 (position c). The GAL4 derivatives were doubly translated in wheat germ extracts as: DBD plus DBD-AR2 (lane 1), DBD plus DBD-ID1-AR2 (lane 2), and DBD-AR2 plus DBD-ID1- AR2 (lane 3).
4 5 immunoprecipitation and SDS-PAGE as for singly translated species, the results shown in figure 15. Again the crosslinker was either released or retained by the presence or absence of (3- mercaptoethanol in the sample loading buffer, respectively. A heterodimer form was observed amongst DBD and DBD-AR2 derivatives, but no heterodimers were observed when one of the two co-translated GAL4 derivatives contained ID1. Even when co- translated, DBD and DBD-AR2 still formed homodimers, as expected, yet DBD-ID1-AR2 formed neither homodimers or heterodimers. ID1, then, appears to prevent the dimerization function of GAL4, impairing its DNA binding ability.
5. ID1 Promotes Multimerization
Since GAL4 derivatives containing ID1 do not appear to dimerize but instead tend to aggregate as large complexes, I set out to determine the makeup of these complexes. I employed a strategy that would potentially form a ladder of partially formed protein complexes. To accomplish this, I added an approximate 10 fold higher concentration of in vitro translated DBD-ID1-AR2 protein than usually used in crosslinking reactions, while gradually decreasing the concentration of DSP crosslinker used. These reactions were then immunoprecipitated and analyzed by SDS-PAGE, again either including or excluding p-mercaptoethanol from the sample loading buffer.
Figure 16 demonstrates the results of the limited DSP crosslinking experiment with wheat germ extracts. The uncrosslinked lanes displayed the DBD-ID1-AR2 monomer of apparent molecular weight of approximately 48 KDa, as observed in figures 13 and 15 as well. On the other hand, as the concentration of crosslinker was increased in the crosslinked lanes, a gradual disappearance of the excess 48 KDa monomer was observed along with the gradual increase of fully formed large protein complexes at the top of the gel. But, the important observation that could be seen in the crosslinked lanes of figure 16, was the appearance of three additional bands that
46 Figure 15. ID1 Prevents the Formation of Heterodimers
Autoradiogram of a crosslinking experiment examining the formation of heterodimers of [35s]-methionine labelled GAL4 derivatives co- translated in vitro in wheat germ extracts. The co-translated GAL4 derivatives were crosslinked with DSP, immunoprecipitated with mAb 5C8-12 to the DNA binding domain of GAL4 and separated by 10% SDS-PAGE either in the crosslinked state without the addition of P-mercaptoethanol (P-Me) to the sample loading buffer (lanes 1-3), or in the uncrosslinked state with the addition of P-mercaptoethanol to the sample loading buffer (lanes 4-6). The co-translated GAL4 derivatives used were: DBD-AR2 plus DBD-ID1-AR2 (lanes 1 and 4), DBD plus DBD-ID1-AR2 (lanes 2 and 5), and DBD plus DBD-AR2 (lanes 3 and 6). The species indicated by the arrows represent: DBD monomers and dimers (positions a and b, respectively), DBD-AR2 monomers and dimers (positions c and f, respectively), DBD-ID1-AR2 momoners (position d), and DBD plus DBD-AR2 heterodimers (lane 3, position e).
4 7 p-Me ... + + +
1 2 3 4 5 6
48 Figure 16. Limited Crosslinking Reveals that ID1 Causes the Formation of Multimers in Wheat Germ Extracts
Autoradiogram of a crosslinking experiment with limited DSP crosslinker examining the formation of protein multimers of the [35s]-methionine labelled, wheat germ expressed GAL4 derivative DBD-ID1-AR2. DBD-ID1-AR2 was crosslinked with DSP, immunoprecipitated with mAb 5C8-12 to the DNA binding domain of GAL4 and separated by 7.5% SDS-PAGE either in the crosslinked state without the addition of p-mercaptoethanol (P-Me) to the sample loading buffer (lanes 1-6), or in the uncrosslinked state with the addition of p-mercaptoethanol to the sample loading buffer (lanes 6- 12). A gradient of DSP crosslinker concentration was used in the reactions: 0.06 mM, 0.125 mM, 0.25 mM, 0.5 mM, 0.75 mM and 1 mM (lanes 1 to 6 and lanes 7 to 12). The species indicated by the arrows represent: DBD-ID1-AR2 monomers (position a), dimers (position b), trimers (position c), and tetramers (position d). This experiment was only performed once.
4 9 1 2 3 4 5 6 7 8 9 10 11 12
50 were not detected in the uncrosslinked lanes. I suggest that these three bands of apparent increasing molecular weights of approximately 100 KDa, 141 KDa, and 186 KDa represent the dimer, trimer, and tetramer forms, respectively, of the DBD-ID1-AR2 protein. The calculated molecular weights of the dimer, . trimer and tetramer forms of the 48 KDa ID1 containing GAL4 derivative are 96 KDa, 144 KDa and 192 KDa, very similar to the molecular weights of the three protein bands immunoprecipitated with GAL4 mAb 5C8-12 observed in figure 16. The intensity of these three protein bands is rather faint compared to that of the intensity of the monomer bands and to that seen for the bands at the top of the gel. This faintness implies that the higher order multimer forms caused by ID1 are favoured, preferentially forming a large multimer complex. The formation of protein multimers by the DBD-ID1-AR2 derivative was also observed with E. coli expressed GAL4 derivatives in a limited DSP crosslinking experiment, similar to that for in vitro translated proteins. The results of the limited crosslinking experiment performed with E. coli expressed GAL4 constructs are shown in figure 17.
The dimer, trimer, tetramer and higher multimer forms of DBD- ID1-AR2 appear to form naturally in solution as they were also observed when in vitro expressed DBD-ID1-AR2 was immunoprecipitated with GAL4 antibody and separated on a gel without p-mercaptoethanol in the sample buffer and in the absence of any DSP crosslinker (data not shown). The same result was also observed with E. co//-expressed DBD-ID1-AR2 (data not shown). These protein multimers probably do not exist normally in solution with wild type GAL4 in yeast as ID1 is kept inactive by the GRD in the absence of glucose, but may form in the presence of glucose when ID1 is active.
The formation of only a small amount of ID1 containing dimers would impair the ability of this protein to bind DNA. This latter observation agrees with the results of the experiments detailed above. The mechanism of action of ID1 of GAL4, then, appears to be
5 1 Figure 17. Limited Crosslinking Reveals that ID1 Causes the Formation of Multimers in E. coli
Autoradiograms of a crosslinking experiment with limited DSP crosslinker examining the formation of protein multimers of the GAL4 derivative DBD-ID1-AR2 expressed in E. coli. DBD-ID1-AR2 was crosslinked with DSP and separated by 7.5% SDS-PAGE either in the crosslinked state without the addition of P-mercaptoethanol (p- Me) to the sample loading buffer (panel A), or in the uncrosslinked state with the addition of P-mercaptoethanol to the sample loading buffer (panel B). A gradient of DSP crosslinker concentration was used in the reactions: 0.06 mM, 0.125 mM, 0.25 mM, 0.5 mM, 0.75 mM and 1 mM (lanes 1 to 6 in each panel). The species indicated by the arrows represent: DBD-ID1-AR2 monomers (position a), dimers (position b), trimers (position c), and tetramers (position d).
5 2 A [DSP]
f ^
-P-Me b+
3 1 2^3 A 5 6
B [DSP]
+ (3-Me
1 2 3 4 5 6 the formation of protein multimers, preventing dimer formation and impairing DNA binding.
6. ID1 Appears to Act by Itself
My results suggest that ID1 causes protein multimerization to prevent DNA binding of GAL4, inhibiting GAL4 function. The limited DSP crosslinking experiment that elucidated the multimerization mechanism suggested that the complexes are homogenous judged from the apparent molecular weights of the multimer bands. Also, G. Stone (1992) had attempted to titrate out a cellular factor or factors that possibly bound to ID1 to inhibit GAL4 activity by overexpressing an ID1 protein fragment in yeast. This titration attempt proved unsuccessful, further suggesting that ID1 imposes inhibition without the aid of other factors. I set out to resolve this aspect of ID1 functioning.
I expressed histidine-tagged versions of GAL4 derivatives in E. coli. These derivatives consisted of his-DBD, his-DBD-AR2 and his- DBD-ID1-AR2. The six histidine residues tag expressed N-terminal to the GAL4 fusions allowed these proteins to be purified with the use of Ni2+-NTA -agarose beads. I purified each GAL4 fusion protein using either a batch or a column purification procedure, looking for a protein or proteins that would be purified along with his-DBD-IDl- AR2 but not with the other GAL4 fusions. If such proteins existed, it would suggest that ID1 requires these conserved cellular proteins that bind to ID1 and contribute to inhibition. The batch purifications are displayed in figure 18, whereas the purified column fractions can be found in figures 19, 20, and 21. Both the batch purification attempt and the column purification procedure, which yielded purer protein, failed to produce one or more proteins that copurified solely with his-BDB-IDl-AR2.
Furthermore, I performed EMSA experiments with comparable amounts of either batch purified (figure 22) or column purified (figure 23) histidine-tagged GAL4 derivatives to determine whether
54 Figure 18. Batch Purification of Histidine-tagged GAL4 Derivatives
Coomassie blue staining of SDS-PAGE gels, 15% (panel A) or 7.5% (panel B), of histidine-tagged GAL4 derivatives expressed in E. coli and purified in a batch procedure. E. coli extracts expressing different GAL4 derivatives were incubated with Ni2+-NTA-agarose beads, washed four times with buffer A, at 30 mM imidazole, then eluted with buffer B, at 250 mM imidazole. Lane 1 in both panels represent the batch purification of the vector control pRSETB, lane 2 in both panels that of GAL4 his-DBD (position a), lane 3 in both panels that of GAL4 his-DBD-AR2 (position b), and lane 4 in both panels that of GAL4 his-DBD-IDl-AR2 (position c).
5 5 56 Figure 19. Column Purification of the GAL4 Derivative his-DBD
Coomassie blue staining of the column fractions 1-24 (panels A, B, and C) containing the purified GAL4 derivative his-DBD separated on a 10% SDS-PAGE gel. The his-DBD protein was expressed in E. coli and loaded on a 2 mL column of Ni2+-NTA-agarose beads. The column was run on an Econo System apparatus at 4°C with a flow rate of 0.5 mL/min with an elution gradient of buffer A at 30 mM imidazole to buffer B at 500 mM imidazole over 1 hour. Samples of the 1 mL column fractions that were collected are shown above. The his-DBD GAL4 derivative is indicated by the arrow a.
57 1 2 3 4 5 6 7 8
910111213141516
1718192021222324 58 1 2 3 4 5 6 7 8
Figure 20. Column Purification of the GAL4 Derivative his-DBD-AR2
Coomassie blue staining of the column fractions 1-8 containing the purified GAL4 derivative his-DBD-AR2 separated on a 10% SDS-PAGE gel. The his-DBD-AR2 protein was expressed in E. coli and loaded on a 2 mL column of Ni2+-NTA-agarose beads. The column was run on an Econo System apparatus at 4°C with a flow rate of 0.5 mL/min with an elution gradient of buffer A at 30 mM imidazole to buffer B at 500 mM imidazole over 20 minutes. Samples of the 1 mL column fractions that were collected are shown above. The his-DBD-AR2 GAL4 derivative is indicated by the arrow a.
59 Figure 21. Column Purification of the GAL4 Derivative his-DBD-IDl-AR2
Coomassie blue staining of the column fractions 1-16 (panels A and B) containing the purified GAL4 derivative his-DBD-IDl-AR2 separated on a 10% SDS-PAGE gel. The his-DBD-ID 1 -AR2 protein was expressed in E. coli and loaded on a 2 mL column of Ni^+-NTA- agarose beads. The column was run on an Econo System apparatus at 4°C with a flow rate of 0.5 mL/min with an elution gradient of buffer A at 30 mM imidazole to buffer B at 500 mM imidazole over 40 minutes. Samples of the 1 mL column fractions that were collected are shown above. The his-DBD-ID 1-AR2 GAL4 derivative is indicated by the arrow a.
60 97 66 45 29
1 2 3 4 5 6 7 8
97 66 45 29
910111213141516
6 1 Figure 22. EMSA of Batch Purified Histidine-tagged GAL4 Derivatives
Equal amounts of the batch purified E. coli derivatives his-DBD (lane 2, position b), his-DBD-AR2 (lane 3, position c) and his-DBD-IDl-AR2 (lanes 4-6, position d) were added to EMSA DNA binding reactions with a GAL4 specific oligo. Arrow a displays the protein breakdown product forming a GAL4 minimal DNA binding domain. The GAL4 oligo alone is shown in lane 1. A 100X excess of unlabelled, specific competitor (sp. competitor) GAL4 oligo was added in the reaction in lane 5, whereas the reaction in lane 6 contained a 100X excess of an unlabelled, non-specific competitor (n. sp. competitor) human RBF protein oligo.
62 sp. competitor n. sp. competitor Figure 23. EMSA of Column Purified Histidine-tagged GAL4 Derivatives
Equal amounts of the column purified E. coli derivatives his-DBD (lane 2, position b), his-DBD-AR2 (lane 3, position c) and his-DBD- ID 1-AR2 (lanes 4, position d) were added to EMSA DNA binding reactions with a GAL4 specific oligo. Arrow a displays the protein breakdown product forming a GAL4 minimal DNA binding domain. The GAL4 oligo alone is shown in lane 1. The column fractions used were fraction 11 for his-DBD, fraction 8 for his-DBD-AR2, and fraction 11 for his-DBD-IDl-AR2, samples of which can be seen in figures 19, 20 and 21, respectively.
64 12 3 4
65 the purer ID1 containing protein would behave differently than a more impure form. A difference in DNA binding behaviour would suggest the loss of a necessary factor that perhaps could not be detected by the protein purification attempts. As had been previously observed in other EMSA experiments, such as figure 9, the ID1 containing derivative was severely impaired, to the order of about 100-fold, in binding ability compared to the other GAL4 fusions; however, no difference was found in his-DBD-ID 1-AR2 DNA binding behaviour in relation to purity. This lack of difference again implies that ID1 impairs DNA binding ability without the help of cellular proteins. The small amount of DNA binding observed in figures 22 and 23 with his-DBD-ID 1-AR2 appears to be specific by the competition reactions performed in figure 22. The amount of his-DBD-ID 1-AR2 E. coli derivatives added to the EMSA reactions of figures 22 and 23 was much higher than that added to similar EMSA reactions with non-histidine tagged E. coli versions of DBD-ID1-AR2 (figure 9) which showed no binding ability at all; I believe that the higher the GAL4 fusion protein concentration, the higher the amount of ID1 containing dimers versus multimers that can be formed, thus the minimal DNA binding observed.
The purification results and the EMSA results with the purified GAL4 fusions agree with the limited DSP multimerization data that ID1 appears to be able to inhibit GAL4 derivatives without the aid of any additional cellular factors. However, how the multimerization mechanism occurs in terms of the exact location of the multimerization contacts and whether the multimers are aberrant are still unresolved questions. Nevertheless, it appears that ID1 inhibits GAL4 activity by forming homogenous protein multimers, preventing dimerization and thus impairing DNA binding, a necessary step to achieve transcriptional activation.
7. ID1 is Homologous to Other Proteins
As Stone and Sadowski (1993) originally noted, the short 320- 412 amino acid stretch found in GAL4 known as ID1 is homologous to
66 other proteins, mainly fungal transcriptional activators, but also to other proteins such as CDC-6 and PDR3. Table 2 summarizes some of the proteins homologous to ID1 as well as their respective function and species of origin. The list of proteins having homology to ID1 is constantly growing as the genome sequencing projects of many organisms are progressing, and new genes are discovered. A computer aided sequence alignment (BLITZ search) between ID1 of GAL4 and some of its homologous proteins is displayed in figure 24. The majority of the conserved residues seen in figure 24 are hydrophobic or polar. On the other hand, there are also several conserved charged residues as well as a few particular amino acids that are conserved in all proteins. Three residues in particular may be especially important for the functioning of ID1 or its regulation. These three residues, marked with an asterix in figure 24, were found to be point mutations (Ser 322 to Phe, Leu 331 to Pro, and Ser 352 to Phe) that inactivate GAL4, by Johnston and Dover (1988). These particular residues, especially Leu 331 and Ser 352 which show good homology to the other proteins and may thus serve conserved roles, could be responsible for protein-protein interactions that either govern the regulation of ID1 through contact with the GRD, or mediate protein multimerization promoted by ID1. Either way, mutations at these residues cause unusual amino acid function that render GAL4 inactive.
Since ID1 has been shown to be modular and to function in different fusion protein locations, it is possible that ID1 forms a globular domain, perhaps consisting of the conserved hydrophobic residues interacting with each other and exposing the conserved charged or polar residues. These conserved charged or polar residues may be involved in protein-protein interactions, forming the protein multimers observed with ID1. Conversely, the conserved hydrophobic residues may be important for forming protein-protein interactions. On the other hand, ID1 may not be globular but unstructured while still able to form protein multimers. Nevertheless, there is a strong possibility requiring further investigation that the sequence similarity between ID1 and other proteins may underlie structural and functional
67 PROTEIN PROTEIN FUNCTION SPECIES
LAC9 Lactose/galactose transcription factor K. lactis
PUT3 Proline utilization transcription factor S. cerevisiae
LEU3 Leucine anabolism transcription factor S. cerevisiae
PPR1 Pyrimidine pathway transcription factor S. cerevisiae
THI1 Thiamine repressible genes regulator S. pombe
NIT4 Nitrogen assimilation transcription factor N. crassa
NIRA Nitrogen assimilation transcription factor A. nidulans
PDR3 Pleiotropic drug resistance regulator S. cerevisiae
CDC6 Initiation of DNA replication S. cerevisiae
YHX8 Putative transcription factor S. cerevisiae
YINO Putative transcription factor S. cerevisiae
YJ16 Putative transcription factor S. cerevisiae
YCZ6 Putative transcrition factor s. cerevisiae
YBOO Putative transcription factor s. cerevisiae
YE14 Putative transcripiton factor s. cerevisiae
Table 2. Proteins Homologous to ID1 of GAL4
The proteins listed above with their respective protein function and species of origin contain a high degree of sequence homology to ID1 of GAL4 according to a computer homology search (BLITZ).
68 GAL 4 SGSIILV TALHLLSRYTQWRQ KTNTSYNFHSFSIRMAISLGLNRDLPSS FSD PDR3 KETIYLILR LFDLC YEHLIQ GCISISNPLENYLQKIK QTPTTTASASL PTSPA PPRl NSQLPLLHRELFLKK YFEPIY GPWNPNIALASDQTGINSAFEIPIT SAFSA HTEPK LEU3 ASVYSV QAFLLYT FWPPLT SSLSADTSWNTIGTAMFQALRV GLNCAGFSKEYASAN NIRA NSKLCTV QALALMS VRE A GCGREGKGWVYSGMSFRMAFDLGLN LESSSLRDLSE NIT4 MS VRE A GCGREAKGWVYSGMSFRMAQDIGLN LDIGSL DE LAC 9 GSTDLTI ALILLTHYVQKMH KPNTAWSLIGLCSHMATSL GLHRDL PN PUT 3 I EVLLLYAFFLQ VA DY TLASYFYFGQALRTCLIL GLHVDSQS DTLSR THI1 LI GLYLQSTIYE KSSFAYFGLAIKFAVAL GLHKNSDDPSL TQN CDC 6 IYSI QAIFMMTIFLQCSA NLKACYSFIGIALRAALKE GLHRRSSIVGPTPIQ ! !## ## #####! !## # !!#!# !! #!#!!##+ ## #GL!+ !#!# !##
6AL4 SSILEQRRRIW W SVYSWEIQLSLLY G RSI QL SQNTISFPSS PDR3 PLSNDLVIS VIHQLPQPFIQSITGFTTTQLIENLHDSFSMFRIVTQMYAQHRKRFAEF PPRl RENVTEKID VCSSVDVPWYDT WETSQKVN MRPIVELPTKFHIPYF F LEU3 SELVNEQIRTWICCNWSQTVASSFGFPAYVSFDYLV ISSIR VPN SKSQVDIPNE NIRA EEIDARRITFWGCFLFDKCW S NYLG RQPQF TTANTSVS NXT4 KEVDARRITFWGCFVFDKCW S NYLG RLPQLPKNTYN LAC 9 S RRV LW W TIYCTGCDLSLE TG R PUT3 YEIEHHR RLW W TVYMFERMLSSK AG LPLSFTDYTIS THI1 SKELRNRL LW SVFCIDRFVSMT TG RR PSIPLEC ISIP CDC 6 DET KKRL FW SVYKLDLYMNC ILG !-## ++# #W#! ! #- !W !#!!#- !#! !#! #S #R ## # ! !!#!##!#
Figure 24. Alignment of Protein Sequences Homologous to ID1
of GAL4
The amino acid sequences of the selected proteins were aligned by a BLITZ search (BLITZ search scores of 60 or higher). The aligned residues were scored as either conserved hydrophobic residues (#), conserved polar uncharged residues (!), conserved acidic residues (-) or conserved basic residues (+) if 50% or more of the residues belonged to one of the categories. If 80% or more of the residues was the same amino acid, the one letter code for this amino acid was indicated. Three point mutants (*) in ID1 of GAL4 were found to inactivate GAL4 protein function.
69 similarity. ID1 may prove to have a conserved mechanism of protein-protein interactions, resulting in multimerization and regulation of protein function.
70 DISCUSSION
1. Glucose Inhibits GAL4 Through Its Central Region
My ribonuclease protection assay fully supports the evidence described by Stone and Sadowski (1993) that an important glucose repression mechanism occurs through the central region of GAL4. This central region is composed of a glucose-responsive domain or GRD, which is able to sense the presence of glucose, arid of three inhibitory domains (ID1, 2, 3) which carry out the inhibition of GAL4 activity. The current model is that, in the absence of glucose, the GRD antagonizes the inhibitory domains, perhaps by a direct interaction with these domains. But, upon addition of glucose, a signal is generated which disrupts this interaction, allowing the inhibitory domains to function. This mechanism appears to work rather quickly, as I have demonstrated and as Stone and Sadowski have shown (1993).
There are still many aspects to be clarified about this glucose repression mechanism. It should be established how the signal is relayed from glucose to GRD, whether there is a signal transduction pathway or whether a factor like glucose itself or a metabolite contact GRD directly. In fact, G. Stone (1992) has shown by a titration experiment that an unknown glucose responsive factor interacts with residues 693-767 of GAL4, within the GRD. Furthermore, it would be interesting to determine whether the phosphorylation sites that lie within GRD, such as Ser 699, are involved in the glucose signal transduction and the interaction with this unknown factor. Also, a detailed mechanism of how GRD and the inhibitory domains interact is necessary to understand how this regulation is achieved. Some missense mutations of GAL4 within residues 320-520, the inhibitory region, have been found to inactivate GAL4 (35). It is possible that these mutations disrupt the interaction between the GRD and the inhibitory domains, resulting in constitutive inhibition. Further characterization of these mutants
7 1 may lead to an informative part of the mechanism of action of the GAL4 central region.
2. ID1 Is a Strong Inhibitory Domain Homologous to Other Proteins
Stone and Sadowski (1993) had established that residues 320- 412 of GAL4, a region termed ID1, had a strong inhibitory effect on transcriptional activation. My in vivo results in yeast reconfirm this function for ID1. The mechanism of action of ID1, though, was still unknown. This mechanism appears to be evolutionarily conserved, since I have shown that ID1 functions not only in yeast, but also in E. coli, in wheat germ extracts, in rabbit reticulosyte lysates (data not shown), and even in mammalian cells (73).
Furthermore, the homology that exists between ID1 and other fungal transcription factors as well as other proteins, seen in figure 24, suggests that the ID1 sequence may form a conserved structural motif with a possible important conserved mechanism of action. There appear to be many conserved hydrophobic and polar residues, as well as some charged and specific amino acids between ID1 and the other proteins. The homology to ID1 is found mostly in other fungal transcription factors such as LAC9, PUT3, LEU3, PPR1, NIRA, NIT4 and THI1 that are also involved in regulating a metabolic response to a particular signal, but also in other proteins such as CDC- 6, involved in initiation of DNA replication, and PDR3, involved in pleiotropic drug resistance.
If indeed the ID1 motif is conserved not only in sequence but also in function, "ID1" in these other fungal transcription factors may also work to promote protein multimerization, inhibiting the particular transcription factor under non-inducing conditions. As for how "ID1" may be regulated in the other transcription factors, it is plausible that each has its own "GRD-like" domain that masks inhibition when the inducing signal is present, but releases "ID1" when the signal is absent. It will be interesting to determine
72 whether these other transcription factors are regulated similarly to GAL4 as far as the "ID1" mechanism of action is concerned. As for the similarity to proteins like CDC-6, which is not a transcription factor, the "ID 1-like" domain may equally serve a regulatory function to inactivate CDC-6 through multimerization once DNA replication is no longer needed during the cell cycle.
3. ID1 Impairs DNA Binding
A transcription factor must first bind DNA to be able to activate transcription. My EMSA and GAL4 biotinylated oligo assay data suggest that ID1 severely impairs the DNA binding ability of GAL4. By preventing DNA binding, ID1 can inactivate GAL4. This ID1 inhibition mechanism agrees with the observation Stone and Sadowski (1993) had made that LexA-VP16-IDl fusions extracted from yeast grown in glucose showed decreased DNA binding abilities to six LexA operator sites in EMSA experiments as compared to fusions extracted from yeast grown without glucose. As well, Hirst and Sadowski (unpublished) observed that ID1 containing fusions expressed in E. coli and assayed on a A, phage repressor reporter displayed no DNA binding ability; this was not true, however, of derivatives containing ID2 and ID3, suggesting that the three inhibitory domains impose inhibition differently. Three independent modes of inhibition as opposed to a single one carried out by three separate domains may increase the stringency and rapidity of the regulation. The mechanisms of action of ID2 and ID3 still remain uncharacterized.
The fact that ID1 can impair DNA binding has strong implications with respect to the fate of GAL4 during glucose repression. As mentioned, GAL4 is stringently repressed in the presence of glucose by several mechanisms. One of the mechanisms acts through the central region of GAL4 itself and occurs rapidly. A quick way to ensure that GAL4 is inactive in the presence of glucose would be by forcing GAL4 off its DNA binding site, preventing GAL4 from further activating transcription of the GAL genes. In the
73 meantime, the other glucose repression mechanisms would also ensure GAL4 inactivation. The URSQ binding repression complex would rapidly act on any GAL4 dimers that are still bound to DNA, while further induction and GAL4 gene transcription are prevented, and GAL80 mediated repression is re-established. An interplay of all these mechanisms would make certain that no GAL gene transcription occurs in the presence of glucose.
My results regarding the impairement of DNA binding exerted by ID1 on GAL4 also suggest another important result. Giniger et al. (1985) have suggested by in vivo footprinting experiments that GAL4 is not bound to DNA in glucose; however, this result was complicated by the fact that GAL4 gene expression is repressed in glucose, questioning the amount of GAL4 protein left in the cell to bind DNA. On the other hand, Lohr et al. (1985) argued that GAL4 remains bound to DNA, regardless of carbon source, by DNAse I hypersensitivity experiments. My results, those of Stone and Sadowski (1993), and of Hirst and Sadowski (unpublished), then, would support the in vivo footprinting data of Giniger et al. (1985), and would argue against the results of Lohr et al. (1985). Since glucose repression upon GAL4 through its central region occurs rapidly, the uncertainty of whether any GAL4 protein remained in the cell is lifted. My results, then, strongly support the evidence that GAL4 is found bound to its UASG site in the absence of glucose, but is removed from the DNA in the presence of glucose.
4. ID1 Prevents Dimerization and Promotes Multimerization
Carey et al. (1989) showed that GAL4 binds to its 17 mer dyad symmetrical site as a dimer. Without dimerization, which occurs partly through residues 65-94, GAL4 cannot bind DNA. My chemical crosslinking results suggest that the presence of ID1 in in vitro synthesized GAL4 derivatives prevents the formation of homodimers. The lack of dimerization would impair DNA binding, as was observed with the DNA binding assays I performed. The mechanism seems specific to the ID1 region because ID1 containing
74 derivatives not only cannot homodimerize, but also cannot heterodimerize. The dimerization contact found in the DNA binding domain of GAL4 is known to be somewhat weak. It is thought that there exists another dimerization domain in the C-terminus of the GAL4 protein (32). Nevertheless, ID1 seems able to disrupt dimer formation. Alternatively, ID1 may promote aberrant dimers and multimers that cannot bind DNA.
The mechanism of disruption of dimer formation by ID1 appears to be the formation of GAL4 multimers, as indicated by the limited chemical crosslinking experiments I performed. By the molecular weights of the multimer species formed, these GAL4 multimers seem to be homogenous. Some dimers were observed with the DBD-ID1-AR2 GAL4 derivative, and some EMSA results with histidine-tagged derivatives indeed suggest that this derivative has a very limited DNA binding potential, probably provided by the small proportion of normal dimers it can form. Nevertheless, it appears that higher order multimers and even monomers are favoured.
It is still unclear, though, where the multimer contacts form and whether the multimers formed are aberrant with respect to the normal dimerization of GAL4 molecules. Assuming that no other factors are involved from the molecular weights of the multimers, my chemical crosslinking results allow the possibility that ID1 multimerizes by contacting intermolecularly either GAL4's DBD, AR2 or ID1 itself. An important control to try would be a DBD-ID1 fusion. If DBD-ID1 also did not dimerize but formed multimers, it would imply that multimerization does not involve ID1-AR2 contacts. On the other hand, Stone and Sadowski (1993) observed that if either AR1 or AR2 is deleted from GAL4, GAL4 is rendered inactive. In this case, it may be possible that ID1, a rather basic region, may be able to interact with either one of the two acidic activating regions, leaving one of them free to function. Upon deletion of either one of the activating regions, the second, being masked by ID1, may be unable to activate transcription. Finally, yet another observation comes from my heterodimerization experiments. Since DBD-ID1-AR2
75 was unable to form heterodimers with either DBD nor DBD-AR2, this result raises the possibility that the multimerization contacts are intermolecular LD1-ID1 contacts. Further experimentation is required to resolve this aspect of ID1 functioning.
5. ID1 Does Not Require Other Cellular Factors
From the molecular weights of the multimers obtained with the limited chemical crosslinking experiments, it appeared that ID1 formed multimers that were homogenous, I pursued this issue further by purifying histidine-tagged GAL4 fusions from E. coli to determine whether a protein or proteins copurified with the ID1 containing derivative but not with the other derivatives. Several proteins copurified with HIS-DBD-ID 1-AR2, but none were unique to this GAL4 derivative purification. Furthermore, the purified DBD- ID1-AR2 derivatives behaved similarly in EMSA experiments, regardless of their level of purity, suggesting that no protein necessary for ID1 functioning was lost. The purification procedures, then, also support the chemical crosslinking results with the idea that ID1 does not require other proteins to function. Another possibility is that ID1 requires a cellular factor other than a protein to cause inhibition. This possibility was discredited by G. Stone (1992) since an attempt at titrating a factor binding to the ID1 region by overexpressing ID1 failed. The models I proposed above for direct multimer contacts between ID1 and DBD or AR2 or ID1 itself, then, seem valid.
6. Comparison of ID1 to Other Known Inhibitory Domains
Many transcriptional activators have been shown to contain regions that lack sequence homology to ID1 but that also cause transcriptional inhibition. The mechanisms of action of these other inhibitory domains are somewhat similar yet different from GAL4 ID1 mechanism of multimerization, impairing DNA binding. For example, the inhibitory POZ domain found in poxvirus transcriptional activators such as ZID, Ttk and GAGA has been described to also
76 prevent DNA binding by promoting protein-protein interactions without the aid of other cellular factors (3). These POZ domain promoted protein-protein interactions are believed to interfere with the interaction of the Cys2-His2 zinc finger DNA binding domain with DNA. The POZ domain mechanism of inhibition is highly similar to that of ID1, but it is still unclear what part of protein activity ID1 multimerization interferes with. On the other hand, the inhibitory domain of transcription factors Ets-1 and Ets-2 causes inhibition of protein function by interfering with DNA binding directly and not homodimerization of Ets-1 and Ets-2 (30), unlike ID1. The opposite is true of the inhibitory domain found in the transcriptional activator E12, which interferes with homodimerization of E12 but not with DNA binding of MyoD-E12 heterodimers (75). Finally, other transcription factors such as Oct-2 and c-Fos contain inhibitory domains that unlike ID1 require other cellular factors to carry out inhibition by titration experiments (10, 48); c-Fos inhibitory domain 1, however, contains some important basic residues as also seems true for GAL4 ID1. ID1 mechanism of action, then, is similar to that of other inhibitory domains, yet unique, representing a new mode of inhibition.
7. Model of the Mechanism of Action of ID1
Figure 25 depicts a summary model of the proposed mechanism of action of ID1 of GAL4. In the absence of glucose, the inhibitory domains of GAL4 are believed to be kept inactive by the interaction, whether direct or through some intermediary factor, with the GRD. In the presence of glucose, a signal is relayed to the GRD, possibly in the form of the titratable glucose-responsive factor mentioned above. The interaction between the GRD and the inhibitory domains is thus disrupted, allowing the inhibitory domains to function. In the case of ID1, this inhibitory domain would cause protein multimers to form, either through ID 1-DBD, ID1-AR2 or ID1- ID1 contacts. These multimers are unable to bind DNA, rendering GAL4 transcriptionally inactive.
77 Figure 25. Proposed Mechanism of Action of ID1 of GAL4
In the absence of glucose (- GLUCOSE), the glucose response domain (GRD) interacts with the inhibitory domains (ID1, ID2, and ID3), either directly or through an intermediary factor, and antagonizes their function. GAL4 is bound to DNA through its DNA binding domain (DBD) and can activate transcription through its two activating regions (AR1 and AR2). Upon addition of glucose (+ GLUCOSE), a signal is generated which causes a glucose-responsive factor (G.R.F) to bind to GRD, freeing the inhibitory domains. ID1 can now act by causing the formation of GAL4 protein multimers, by contacting either DBD, ID1 or AR2 intermolecularly (possibility 1, 2 or 3, respectively). These protein multimers are unable to bind to DNA, rendering GAL4 transcriptionally inactive.
78 - GLUCOSE
ADO GRD ID3 INTERACTION D DBD AR1 ID1 1 i r
+ GLUCOSE GLUCOSE
DBD AR1 ID1 ID2 ID3 GRD AR2
GAL4 PROTEIN MULTIMERS
ID1-DBD ID1-ID1 ID1-AR2 CONTACTS CONTACTS CONTACTS REFERENCES
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