78-5907

PHILLIPS, Steven John, 1949- LEUCINE TRANSFER RNA AMINOACYLATION IN TWO STRAINS OF BACILLUS SUBTILIS DURING BALANCED GROWTH AND LEUCINE STARVATION.

The Ohio State University, Ph.D., 1977 Microbiology

University Microfilms International,Ann Arbor, Michigan 48106 l e u c i n e t r a n s f e r rna aminoacylation in two

STRAINS OF EACILLUS SUCTILIG DURING BALANCED GROWTH

AND LEUCINE STARVATION

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of the Ohio State University

i->y0 ,»

Steven John Phillips, 13. A., M. S.

The Ohio State University

1977

Reading Committee:

Dr. James C. Copeland Appr oved by Dr. Thomas J. Byers

Dr. Robert M. Pfister ' M u i - Adviser

Department of Microbiology DEDICATION

To my family ... and to my lady.

ii ACKNOWLEDGEMENTS

I would like to thank my adviser, Dr. James C. Copeland, for his guidance and instruction over the course of our seven year association as adviser and student. I would also like to thank my reading committee, Dr. Thomas J. Byers and Dr.

Robert M. Pfister, for 'their extraordinary efforts with respect to the completion of this dissertation in final form.

I thank Dr. Donald Dean and Dr. George Marzluf for their advice and comments. I thank Mr. Ralph T. Rogers for the figures in this dissertation; Ms. Kris Boggs and Ms. Peggy

Chivington for typing the tables; Mr. Ray Fatig for his bibliographic efforts; and Mr. Mark Rudinski for his help in proofing the manuscript. Mr. Michael Peasley performed a number of the experiments on aminoacylation over the course of this past summer with a skill and attention to detail not normally expected of an undergraduate student. His help contributed substantially to the completion of this work.

Mr. Aaron Supowit of IRCC composed a computer program for liquid scintillation counting of Sepharose 4B effluents.

Finally, I thank two good friends — Peggy Chivington and

Mark Noyman -- for enriching my extra-curricular life in special ways. VITA

August5, 1949 Born--Chicago, Illinois

1967 Graduated--Larkin High School, Elgin, Illinois

1971 B. A. (Chemistry), Knox College, Galesburg, Illinois

1974 M. S. (Microbiology) The Ohio State University, Columbus, Ohio

1972-1977 Graduate Teaching and Research Associate, Department of Microbiology, The Ohio State University

PUBLICATIONS

1.Phillips, S. J. and J. C. Copeland. 1974. Effects of single amino acid starvations on chromosome configuration in

Bacillus subtilis. Microbial Genetics Bulletin. 37: 6 -8 .

2. (Abstract) Copeland, J. C. and S. J. Phillips. 1974.

Regulation of chromosome replication in Bacillus subtilis.

Genetics 77: SI3.

3. Copeland, James C., Steven J. Phillips, and Michael W. H.

Mao. 1976. Effect of amino acid starvation on chromosome replication in Bacillus subtilis. In, Microbiology, 1976.

American Society for Microbiology. 70-82. TABLE OF CONTENTS

PAGE

DEDICATION...... ii

ACKNOWLEDGEMENTS...... iii

VITA ...... iv

LIST OF TABLES ...... vii

LIST OF FIGURES ...... viii

INTRODUCTION ...... 1

LITERATURE REVIEW...... 2

Transfer RNAs: Primary Structure...... 2 Transfer RNAs: Secondary Structure ...... 5 Transfer RNAs: Tertiary Structure ...... 6 Transfer RNA Biosynthesis: Transcription...... 7 Transfer RNA Biosynthesis: Processing...... 8 Post-Transcriptional Modification of t R N A ...... 9 Transfer RNA and Protein Synthesis ...... 12 Transfer RNA and Non-Ribosomal Reactions...... 13 tRNA Variability Under Varying Physiological Conditions ...... 15 Viral Induced Changes in thetRNA Pattern 21 tRNA Regulation ofBiosynthetic Pathways ...... 2 3 Rationale for Studying LeucineStarvation 30 Transfer RNA: Effect of Amino Acid Stair vat ion ...... 31

MATERIALS AND METHODS...... 45

Bacterial Strains ...... 45 Media ...... 45 Chemicals ...... 50 Column Materials ...... 50 Radioisotopes ...... 50 Enzymes ...... 50 Preparation of Aminoacyl tRNA Ligase (tRNA Synthetase) ...... 50

v TABLE OF CONTENTS (continued)

PAGE

MATERIALS AND METHODS (continued)

Starvation Regimen ...... 52 Preparation of Transfer R N A ...... 54 Determination of Amino Acid Acceptor Capacity ...... 55 Periodate Treatment of t R N A ...... 56 Sepharose 4B Chromatography ...... 58 Chemical Determinations of DNA ...... 59 Chemical Determinations of RNA ...... 60 Chemical Determinations of Protein...... 60 Sucrose Density Gradient Analysis ...... 61

RESULTS ...... 63

Growth Kinetics and Viability...... 63 DNA Stoichiometry ...... 64 Preparation of tRNAs: Retention of Aminoacyl Ester s ...... 64 Sucrose Gradients ...... 71 Determination of Acceptor Activity: Base Hydrolysis ...... 81 Kinetics of Leucine Incorporation ...... 82 RNase Control ...... 85 Periodate Treatment ...... 89 Kinetics of Periodate Oxidation ...... 94 Aminoacylation Levels ...... 96 Sepharose 4B Chromatography of Leucyl tRNA ...... 99

DISCUSSION ...... 122

Periodate Treatment ...... 122 Bulk Aminoacylation Levels ...... 124 Sepharose 43 Chromatography ...... 134

SUMMARY ...... 142

APPENDIX ...... 144

A ...... 144 B ...... 149

BIBLIOGRAPHY...... 154

vi LIST OF TABLES

TABLE PAGE

1. Strains of Bacillus subtilis Used In This Study ...... 46

2. Effects of Amino Acid Starvations ...... 67

3. RNA Concentration and Tritium Activity Before and After Isolation ...... 69

4. Percentage of 4_S RNA Relative to Total RN A ...... 80

5. Sensitivity to RNase ...... 88

6 . Effects of Various Agents on the Aminoacylation Reaction...... 91

7. Kinetics of Periodate Oxidation ...... 95

8 . BCN503 Aminoacylation Levels ...... 97

9. BC29 Aminoacylation Levels ...... 100

10. Sepharose 4B Chromatography Summary...... 120

11. Aminoacylation Levels in BCK'503 Experiment1 ...... 145

12. Aminoacylation Levels in BCW503 Experiment2 ...... 146

13. Aminoacylation Levels in BCW503 Experiment3 ...... 147

14. Aminoacylation Levels in BCW503 Experiment4 ...... 148

15. Aminoacylation Levels in BC29 Experiment1 ...... 150

16. Aminoacylation Levels in BC29 Experiment2 ...... 151

17. Aminoacylation Levels in BC29 Experiment3 ...... 152

18. Aminoacylation Levels in BC29 Experiment 4 ...... 153

vi i LIST OF FIGURES

FIGURE PAGE

1. Structure of tRNA ...... 3

2. Representation of a genetic map for Bacillus subtilis ...... 48

3. Growth kinetics for BC29 and BCW503 when starved for the amino acid leucine...... 65

4. Sucrose gradient analysis (A254) of RNA isolated from BC29 in balanced growth...... 72

5. Sucrose gradient analysis (A254) of RNA isolated from BC29 starved for leucine for two generation time equivalents...... 74

6 . Sucrose gradient analysis (A254) of RNA isolated from BCW503 in balanced growth...... 76

7. Sucrose gradient analysis (A254) of RNA isolated from BCW503 starved for leucine for two generation time equivalents...... 78

8 . Kinetics of base hydrolysis of aminoacyl-tRNA..83

9. Kinetics of leucine incorporation into untreated and base-hydrolyzed tRNA...... 86

10. Sepharose 4B chromatography of leucine tRNA aminoacylated with inactive synthetase...102

11. Sepharose 4B chromatography of leucine tRNA isolated from cells of BC\^!503 in balanced growth...... 106

12. Sepharose 4B chromatography of leucine tRNA isolated from cells of BCW503 after leucine starvation for two generation time equivalent ...... 109

viii LIST OF FIGURES (continued)

FIGURE PAGE

13. Sepharose 4B chromatography of leucine tRNA isolated from cells of BC29 in balanced growth...... 112

14. Sepharose 4B chromatography of leucine tRNA isolated from cells of BC29 in exponential growth...... 114

15. Sepharose 4B chromatography of leucine tRNA isolated from cells of BC29 after leucine starvation for two generation time equivalents...... 117

ix INTRODUCTION

Amino acid starvation has been used for the last three decades to arrest protein synthesis in bacteria. The simplest explanation for this arrest of protein synthesis is that during starvation, the previously aminoacylated (charged) transfer ribonucleic acids (tRNAs) are exhausted and con­ sequently a large pool of uncharged tRNAs accumulates in the cell.

In spite of the fact that many workers have used amino acid starvation to arrest protein synthesis, only a few studies have been done to determine the actual state of charging of the tRNAs in the cell during amino acid star­ vation. The object of this study is to determine the relative states of charging of Bacillus subtilis leucyl tRNA during balanced growth and during leucine starvation. Furthermore, individual isoaccepting transfer RNAs (tRNAs with different primary structures that can be aminoacylated by the same amino acid) have been fractionated on Sepharose 4B columns to determine the relative changes in aminoacylation of separable leucine-tRNAs. LITERATURE REVIEW

Transfer RNAs; Primary Structure

Holley et al. (1965) were the first to determine the complete sequence of a natural polynucleotide, tRNA-ala, from baker's yeast. To date over seventy five tRNA sequences have been determined (Rich and RajBhandary, 1976).

The tRNA polynucleotide is an unbranched chain ranging from 75 to 85 nucleotides in length. A chairacteristic peculiar to tRNAs is the high proportion of modified nucleo­ tides. These modified nucleotides include alkyl derivatives of the common nucleotides as well as dihydro- and thio- uridylic acids and pseudouridylic acid (psi). While the absolute sequences vary between tRNAs of the same organism, and even among isoaccepting tRNAs, some structural features are common to all tRNAs. The clover leaf model of Holley is the most convenient representation of the common features

(Figure 1).

This model consists of five arms with the descriptions amino acid, TpsiC (ribothymidine, pseudouridine, cytosine), dihydrouracil (D loop), and extra. The first arm has an unpaired sequence of four nucleotides 3'OH-ACCA- (A: adenine,

C: cytosine) called the tail. The four other arms can each be divided into a complementary helix and an unpaired loop section. 3

Figure 1.

Structure of t-RNA with the nomenclature for the dif­

ferent regions. 4

AMINO ACID

AMINO ACID ARM (7 BASE-PAIRS) TYC HELIX (5 BASE PAIRS) DHU HELIX (3-4 BASE-PAIRS) TYC LOOP (7 NUCLEOTIDES) DHU LOOP (8-12 NUCLEOTIDES)

ANTICODON HELIX (5 BASE-PAIRS) EXTRA ARM (VARIABLE LENGTH)

PYRIMIDINES MODIFIED PURINE

ANTICODON LOOP (7 NUCLEOTIDES)

WOBBLE BASE ANTICODON

fig u re 1 The amino acid, TpsiC and anticodon arms are all of con­ stant length and the length of their helix and loop regions are equal. The dihydrouracil helix contains three or four base pairs and a loop region of eight to ten nucleotides. The extra arm is much more variable and in some tRNA species con­ tains no helix at all. The variation in tRNA sizes from 73 to 93 nucleotides in length is accounted for by variations in these two regions -- the dihydrouracil arm and the extra arm.

As a rule, the helices contain more GC (guanine:cytosine) than AU (adenine:uracil) pairs. This higher proportion of GC pairs may act to stabilize the relatively short helical regions since GC bonds are stronger than AU bonds. In many of the helices there is one site where a non-standard base pair occurs. Often this pairing is GU. Other uncommon base pairs are GA in wheat germ tRNA-phe; UU in baker's yeast tRNA-ala; and CA in Escherichia coli tRNA-fmet. In many tRNAs the anticodon helix contains a pseudouridine: adenine pair.

Transfer RNAs: Secondary Structure

As a first approximation, it seems likely that most of the base pairs in transfer RNA will be of the standard

"Watson-Crick" nature as AU or GC. Unusual base pairs, such as those mentioned in the previous section would be expected to disturb the regular helix. The occurrence of an AC pair in tRNA-fmet may be associated with this tRNA's special func­

tion as the initiator tRNA. Arnott (1971) has postulated

that the relative frequency of the unusual GU pair may occur

by G adopting an unusual tautomeric form so that a standard

Watson-Crick pair can occur.

Arnott et al. (1968) have demonstrated that the confor­

mation of RNA complementary helices varies only slightly over

a wide range of nucleotide compositions. Consequently, there

is good reason to assume that in regions of extensive base pairing, the tRNA adopts a double helical conformation with

either an 1 1 -fold (low ionic strength) or a 1 2 -fold (higher

ionic strength) helix.

Transfer RNAs: Tertiary Structure

At present, at least eight models for the tertiary

structure of tRNA molecules have been proposed (reviewed in

Arnott, 1971). Arnott has pointed out that any model of tRNA

structure must incorporate many or all of the following

general properties:

1. A substantial portion of the molecule should have the same shape in all tRNAs in order that the tRNA can inter­ act in a regular manner with- the ribosome. Further, this common shape should allow close juxtaposition of the -ACCA tails of two tRNA molecules each bound by their anticodons to the appropriate triplet of a messenger RNA molecule.

2. The molecule should be energetically stable, so that non-polar groups are stacked "within" while polar groups are accessible to water.

3. There should be no excessive deviation of bond lengths and angles from those shown to be present in synthetic polymers. 7

4. Helical regions should show a periodicity of 11 or 12 bases.

5. There should be approximately 25 base pairs and about 80 hydrogen bonds per tRNA molecule as shown by optical rotatory dispersion and hydrogen exchange experi­ ments (Cantor et al., 1966).

6 . The axial ratio of the tRNA molecule should be 4:1 characteristic of a long thin molecule.

7. The shape of the molecule should readily account for the regular periodicities observed in tRNA crystals.

8 . The anticodon and extra arms should be exposed (Brostoff and Ingram, 1967) as well as the cytosines of the dihydrou'racil loop (Cashmore e^t aT. , 1971).

9. The TpsiC loop should be inaccessible to polar solvents (Nelson and Holley, 1967; Yoshida and Ukita, 1966).

10. Yaniv and Barrel (1969) have observed photoreactions involving the thiouridine residue at position 8 and the cytidine residue at position 13 in several E. coli tRNAs. This photoreactivity is taken to indicate the close spatial association of these two residues.

11. Each tRNA contains four or five clearly double helical regions.

In 1975 the results of the 2.5A® analysis of yeast tRNA-phe were published (Quigley e^t aT. , 1975; Ladner et_ al. , 1975).

The molecule is somewhat flattened and "L-shaped." The

TpsiC arms form one limb and the D-arm and anti-codon arm form the other.

Transfer RNA Biosynthesis: Transcription

In vitro studies of tRNA transcription using tRNA genes from ^80psuIII+ (a plaque forming phage of E. coli carrying the gene for tyrosine tRNA with a G to A substitution at position 25) DNA and purified E. coli RNA polymerase have demonstrated that the RNA transcript is larger than the mature tRNA. This suggests that the primary transcript is a pre­ cursor molecule whose maturation is brought about by partial degradation (Daniel et_ al. , 1970; Ikeda, 1971). Both sigma factor (the RNA polymerase initiation factor, Littauer et al. ,

1971) and rho (the RNA polymerase termination factor, Ikeda,

1971) appear to be involved in the synthesis of tRNA. In vivo labelling experiments (Bjork, 1975) using an E. coli mutant defective in the synthesis of 5-methyluridine have identified phage T4 precursor tRNA molecules. Gel filtration showed them to be more than twice the size of mature tRNA.

Transfer RNA Biosynthesis:__Processing

Altman (1971) and Altman and Smith (1971) have isolated precursor tRNA-tyr molecules from E. coli cells infected with

^580psujjj. One of these precursors (band Y) has been com- < pletely sequenced. It consists of a molecule 129 nucleo­ tides long containing the complete tRNAsujxi-tyr including the 3'-CCA sequence. This complete tRNA suji j "iy* is bracketed by a 41 nucleotide segment at the 5'-terminus and a three nucleotide sequence at the 3’-OH end. The 5 ’-terminal residue is pppGp which indicates that this is, in fact, the in,vivo transcript. A ribosome bound nuclease (RNase P) removes the 41 nucleotide 5'-sequence intact, exposing the 9

5* end of the mature tRNA (Robertson ejt aJ. , 1972). The 3' end of the precursor tRNA is not attacked by the nuclease, but it is cleaved by another RNase, RNase Q (Perry, 1976).

Post-Transcriptional Modification of tRNA

Precursor tRNA molecules as transcribed are deficient in modified bases. Modification, therefore, takes place at the polynucleotide level. tRNA modifications include methyl- ation (Starr and Sells, 1969), thiolation (Lipsett, 1972), isopentenylation (Hall, 1970), 2 -thiomethyl-6 -isopentenyl adenosine (2MSiPA) formation (Gefter, 1969), pseudouridine formation (Johnson and Soil, 1970), ester ification of

5-carboxymethyluridine (Bronskill et al., 1972) and the direct incorporation of threonine into the N-(purin-6 -yl) carbamoyl threonine residue of rat liver and E. coli tRNA

(Chheda ert al. , 1972).

Methylation has been the most extensively studied of the tRNA modifications. This is due to the relative ease with which the reaction can be followed using methyl-radio­ labelled methionine and to the ease with which methyl defi- - cient tRNA can be obtained. Bjork (1975) has mapped the trmA gene responsible for the production of 5-methyluridine in E. coli. It is located at 79 minutes between argH and rif. It shows 65 percent co-transduction with argH. Methyl deficient tRNA is usually obtained by starving relaxed cells for methionine (Starr and Sells, 1969). The tRNA obtained from these cells is a mixture of normally methylated and methyl deficient tRNA. This does not mean that methionine must be the methyl donor. Kgellin-Strarby

(1969) and Kjellin-Strarby and Phillips (1969) have shown that methyl deficient tRNA accumulates only in Met- yeast that accumulate S-adenosylhomocysteine, a known tRNA methylase inhibitor. Yeast methionine auxotrophs that do not accumulate

S-adenosylhomocysteine during methionine deprivation also do not accumulate methyl deficient tRNA.

Protein tRNA methylase inhibitors have been isolated from adult rabbit liver and adult rat liver (Kerr, 1970;

Chaney et al., 1970; Kerr, 1971). Kerr (1970) has shown these methylase inhibitors to be lacking in fetal rabbit liver, Novikoff hepatoma, and Morris hepatoma and Ehrlich ascites cells; all of which show high endogenous tRNA methylase activities. Chaney et_ a_l. (1970) have obtained similar findings with Walker-256 breast carcinoma -- a highly malignant breast carcinoma.

Biezunski eit al. (1970) and Marmor et^ al_. (1971) have found no differences in the amino acid accepting capacity of methyl deficient or normally methylated tRNA, in contrast to the earlier results of Shugart et^ al. (1968). Marmor and co-workers examined the coding properties of normal and undermethylated tRNA-met. They found no differences in the 11

initiation factor stimulated binding of tRNA-met to the appropriate codon, nor in the rate of formylation of tRNA-met. However, methyl deficient tRNA-met was acylated more slowly than normally methylated tRNA-met giving some indication that methylation may be important in the inter­ actions of tRNA molecules with the appropriate synthetases.

Furthermore, in a mixed population experiment, Bjork and

Neidhardt (1975) showed that cells lacking fully methyl­

ated tRNA due to a mutation in the trmA gene, were at a disadvantage to cells containing fully methylated tRNA.

Methylation differentially affects the coding properties of tRNA-leu and tRNA-phe. Methyl deficient tRNA-phe binds to ribosomes less efficiently than normally methylated tRNA-phe, but the codon response of either tRNA-phe species is identical (Stern et_ al. , 1970). This is not the case with methyl deficient leucyl-tRNA. Normal, fully methylated

E. coli tRNA-leu responds equally efficiently with poly UG or poly UC. Methyl deficient tRNA-leu reacts more effi­ ciently with poly UC than with poly UG (Capra and Peterkof-

sky, 1966). The increased response to poly UC in these preparations is associated with a chromatographic species of tRNA-leu that migrates similarly to methyl deficient tRNA-

leu (Peterkofsky, 1964). Capra and Peterkofsky (1968) have

shown that the species of tRNA-leu that normally recognizes 12 poly UG exhibited a changed codon response to poly UC when the tRNA was methyl deficient.

The biosynthesis of transfer RNA is, therefore, a multi- step process involving a primary RNA transcript that is subsequently cleaved and then chemically modified. This multiplicity of steps suggests a multitude of sites at which regulation of tRNA synthesis could occur. In addition, as will be discussed in the section on regulation of bio­ synthetic pathways, alterations in the biosynthesis of tRNA- his can cause regulatory alterations in the biosynthesis of other molecules and, indeed, can affect an entire biosyn­ thetic pathway.

Transfer RNA and_ Protein Synthesis

Transfer RNA is the smallest of the macromolecular components associated with protein synthesis. A single organism may contain 40 or more species of tRNA. During protein synthesis, an activated amino acid (Hoag land-e^t al. ,

1968) is transferred to one of a given set of isoaccepting tRNAs by a specific aminoacyl tRNA synthetase. Studies of

E. coli arginyl-tRNA synthetase (Craine and Peterkofsky,

1976) suggest that the enzyme possesses a site for free metal (Mg2+, Mn2+, Co2+, or Fe2+). The binding of one atom of metal lowers the Km for the three substrates -- arginine, tRNA-arg and Mg'ATP -- and increases the Vmax of the reac­ tion. Transfer takes place in such a way that the amino acid 13 carboxyl group esterifies at the 3*-hydroxyl end of the terminal adenosine common to all tRNA sequences. In this aminoacylated form, the tRNA interacts with the ribosome and other macromolecular components of the protein syn­ thesizing apparatus. The ribosome possesses two tRNA binding sites (Warner and Rich, 1964), an aminoacyl tRNA binding site and a peptidyl tRNA binding site.

In bacteria, the initiating tRNA, formylmethionyl-tRNA

(tRNA-fmet) enters the peptidyl site directly (Bretscher,

1966). When the second aminoacyl tRNA has bound to the amino site, its amino acid is covalently bound to the preceding amino acid and the di-peptidyl tRNA moves into the peptidyl site from which the discharged tRNA-fmet has dissociated.

A third aminoacyl tRNA can then enter the vacated aminoacyl site and the process continues until a chain terminating triplet (UAG, UAA or UGA) is reached. To participate in protein synthesis, tRNAs must possess unique structures for interacting with their specific synthetases and common features for ribosome binding.

Transfer RNA and Non-Ribosomal Reactions

Besides their role in protein synthesis, aminoacyl tRNAs can transfer their cognate amino acids to acceptor molecules in non-ribosomal reactions (Soffer, 1974). These reactions are mediated by aminoacyl-tRNA transferases. These non- ribosomal reactions are divided into three types depending on 14 the nature of the acceptor molecule. If the acceptor mole­ cule is an intact protein, the amino acid is added to the

N-terminus of the protein (Soffer, 1973). The enzyme can catalyze the addition of the amino acid to phosphatidyl glycerol to produce an aminoacyl ester (Gould and Lennarz,

1970). Aminoacyl esters of phosphatidyl glycerol are com­ ponents of the cell membrane. The third class of reactions is important in the synthesis of bacterial cell walls. Here, the acceptor is an N-acetyl muramyl peptide (Strominger,

1970) and the tRNAs used in these reactions are somewhat specialized.

Roberts (1972) has identified two glycyl-tRNAs from

Staphylococcus epidermis which do not function in protein synthesis, but do participate in peptidoglycan synthesis.

These tRNAs differ in a number of ways from other tRNAs.

First, they contain only a single modified base, 4-thio- uridine; second, their dihydrouridine loops lack the GG sequence commonly found in other tRNAs; third, one of the tRNA species (IB) contains opposing uridines in what is normally the first base of the dihydrouridine arm.

The anticodons of both species contain the glycine anticodon UCC, however as stated above, they do not parti­ cipate in protein synthesis, probably due to a lack of modi­ fications in the TpsiC loop. The two tRNAs differ by only six base changes and the presence of an additional cytosine in the TpsiC loop of glycyl-tRNA-IB. The discovery by Harada et^ aT. in 1975 that transfer

RNA plays a role as a primer in the synthesis of Rous sarcoma

DNA opened the door on an exciting new role for transfer RNA.

It had been known for some time (Canaani and Duesberg, 1972) that the initiation of Rous sarcoma DNA synthesis Jn vitro required a primer RNA (termed Spot 1 RNA) that sedimented at 4S. Sawyer et |T. (1974) had shown that this same RNA could be isolated from uninfected cells. In 1975 these same authors (Harada et_ al_. ) showed that Spot 1 was identical to host tRNA-trp. In contrast, murine leukemia virus uses tRNA-pro as a primer for DNA synthesis (Rich and RajBhandary,

1976). Transfer RNA in RNA tumor viruses is the subject of a recent review by Waters and Mullin (1977). tRNA Variability Under Varying Physiological Conditions

Transfer RNAs can be resolved into their individual iso-accepting species by a number of chromatographic methods, some of which are discussed below. Resolution of a mixture of transfer RNA isoacceptors into its components yields a pattern of accepting activity, usually with fraction number as the abscissa and some measure of tRNA (e^£. absorbance at 260 nanometers or amount of radiolabelled amino acid incorporated as the ordinate. This pattern is referred to as a tRNA profile.

Alterations in tRNA profiles may result from changes in the levels of tRNA species as well as from a number of other changes including tRNA conformation, synthetase specificity, 16 or post-transcriptional modifications. The existence of multiple isoaccepting species of several tRNAs has led to speculation as to the regulatory roles these tRNAs may play.

The alteration of chromatographic patterns of tRNA of

subtilis during sporulation has been a subject of study for many years.(Sueoka and Kano-Sueoka, 1970). Void (1970) and Chuang and Doi (1972) have demonstrated the presence of two tRNA-lys species (designated tRNA-lys-1 and tRNA-lys-2) in vegetative or sporulating cells. Transfer RNA-lys-1 recognizes the codons AAA or AAG with equal efficiency while tRNA-lys-2 recognizes AAG more efficiently than AAA (Chuang and Doi, 1972). Transfer RNA-lys-2 is not detectable in spores. The two tRNA species are transcribed from different genes (Chuang et_ al_. , 1971) but are identical at their

3 '-terminal oligonucleotide sequences.

Inorganic iron has been shown to affect the tRNA com­ position of E. coli. Growth of cells in low iron medium causes an alteration in the chromatographic properties of five tRNA species accepting leucine, cysteine, serine, tyrosine and phenylalanine (Wettstein and Stent, 1968;

Rosenberg and Gefter, 1969). Rosenberg and Gefter have shown that, at least in the case of tRNA-tyr, iron defi­ ciency reduces the synthesis of the 2 -methylthio group on the isopentenyladenosine residue adjacent to the anticodon.

Cysteine starvation in E. coli results in the production of 17 tRNA lacking 4-thiouridine. Sulfur deficient tRNA-ile isolated from these cells shows an increased acceptance for isoleucine (Harris et al.» 1976).

Alterations in the ratio of two isoaccepting species of tRNA-tyr (tRNA-tyr-1 and tRNA-tyr-2) have been shown to occur in "early" versus "late" exponential phase (Gross and

Raab, 1972). It is interesting to note that it is at the transition point from log phase to stationary phase that sporulating cells begin their commitments to sporulation and that competence occurs maximally. The tRNAs of other organ­ isms might be expected to change as these organisms approach a shift in growth. Indeed, Dejesus and Gray (1971) have studied changes in tRNA species of Rhodopseudomonas spheroides.

These cells can grow in the dark aerobically or anaerobically in the presence of light to allow for bacterial photosyn­ thesis. These authors have observed increases in the minor species of tRNAs for tryptophan and phenylalanine when anaerobic photosynthetic cells acre placed under aerobic conditions in the dark.

Transfer RNA populations within cells have also been shown to be altered in cells committed to massive synthesis of specific proteins; in embryonic versus adult tissue; in cells stimulated by hormones and in malignant cells.

Littauer and Inouye (1973) provide an excellent summary of work done until 1973 on such topics as tRNA levels in specific 18 tissues, levels during erabryogenesis, and hormonal regu­ lation of tRNA. A more current, though less comprehensive review is that of Rich and RajBhandary (1976). A recent review by Clark (1977) summarizes the known structural features of tRNA and attempts to correlate them with its biological functions.

Mukerjee and Goldfeder (1976) compared the histidyl, lysyl, tyrosyl, and phenylalanyl transfer RNAs of normal mouse mammary gland and liver with those of three mouse mammary tumors: DBA-g, DBAH, and DBAf^. Methylated albumin kieselguhr (MAK) chromatography revealed extra isoaccepting

species of some tRNAs of the tumors. DBA3 possesses altered lysyx and histidyl-tRNAs. DBAH possesses altered

tRNA-lys and tRNA-phe. DBAH2 exhibits one altered tRNA — tRNA-phe. No differences were observed for the number of isoacceptors of tRNA-tyr. The level of acylatable tRNA-lys of tumor, and liver are identical. In contrast, increases were observed for the level of acylatable tRNA-tyr and tRNA-his in DBA3 ; tRNA-tyr and tRNA-phe in DBAH and only for tRNA-phe in DBAH2 . This study extends the list of differences in specific tRNAs between normal and neoplastic cells. This list includes Ehrlich ascites and virus-trans­ formed cells (Taylor et al., 1968), Morris hepatoma (Srini- vasan et al., 1971), Novikoff hepatomas (Baliga et^ aT., 1969) and human lymphocytic leukemia cell line (Gallo, 1969). 19

There is no change in the transcription of tRNA genes nor in the level to which tRNA is aminoacylated dur ing the growth or development stages in Dictyostelium discoideum

(Palatnik and Katz, 1977). However, changes are detected in the elution profiles for isoaccepting tRNAs isolated at each stage, indicating variability in the amount of trans­ criptional modification between the two stages.

Shenoy and Rogers (1977) tested the hypothesis that dietary amino acids control protein synthesis in rat liver through changes in either the level of tissue amino acids or of charging of tRNA. They investigated the transfer RNA charging levels and total amino acid accepting capacity in the liver of fed or 48 hour starved rats. Under these conditions, the liver decreases 34 to 49 percent in mass.

The hepatic concentrations of eighteen amino acids were measured and no differences were found. The concentrations observed were well above the known Km values of the cognate synthetases, indicating that during starvation the liver maintained a sufficiently high concentration of amino acid to charge tRNA near the Vmax. Their periodate resistance data show that during starvation, there is an 11.4 percent increase in periodate protection of tRNA-phe. Aminoacylation levels for tRNA-ile, tRNA-trp and tRNA-leu decreased respectively 2.2, 10.2 and 8 . 8 percent. During starvation the total acceptor capacity per mg tRNA remained unchanged.

This indicated a decrease in the total amount of biologically 20 active phenylalanyl-tRNA. These results suggest that in the liver the mechanism of regulation of protein synthesis is not mediated through a decrease in the concentration of required amino acids and that the reduction in protein synthesis observed during starvation is due, at least in part to a reduced total amino acid acceptor capacity of the liver cell sap.

The results of Shenoy and Rogers (1977) contrast with those of Fournier et_ al^. (1976) who showed that the half- lives of five tRNA species of Bombyx mori are constant between two distinct physiological states of the organism.

The two states studied were the growth and the secretion phases. The authors examined the synthesis rates and half- lives of five tRNAs in the posterior silk gland. The half- lives were: 70 hours for tRNA-thr; 92 hours for tRNA-ala;

97 hours for tRNA-gly; 100 hours for tRNA-asp and 130 hours for tRNA-ser. The rates of synthesis of each of these species varied widely in direct proportion to the amount present at that phase. Thus, tRNA species that occur at high levels in a given physiological state are synthesized more rapidly than those species present at lower levels.

The variation in tRNA levels seen in this organism at dif­ ferent states of growth results from regulation of tRNA at the transcriptional, processing or modification levels.

This rules out a mechanism of regulation through selective degradation of mature tRNA species. Zilberstein et al. (1976) studied the function of a minor species of leucyl tRNA in normal mouse L cells. Cul­ tures of mouse cells treated with interferon yield extracts that have lost their ability to translate exogenous mes­ senger RNAs (Falcoff et al., 1973). Subsequent translation is dependent on the addition of tRNA from untreated cells

(Content et_ al., 1974). Zilberstein and cj-workers purified three minor tRNA-leu species required for exogenous RNA translation. These minor tRNA-leu species represented about five percent of the total tRNA-leu population. The minor species could not be replaced by synonymous major species. Thus, the minor tRNA-leu species act directly to control translation in normal cells.

Viral Induced Changes in the tRNA Pattern

Many viruses induce changes in the translational machinery of the host. Among these are modifications of the host aminoacyl-tRNA synthetases (McClain et^ a_l. , 1975;

Comer and Neidhardt, 1975) and changes in the tRNA profiles of infected cells due to production of phage coded tRNA

(Sue oka and Kano-Sueoka, 1970; Daniel et^ al_. , 1970b;

Littauer et_ al_. , 1971a; Bjork, 1975).

Infection of E. coli by bacteriophage T4 leads to production of a phage coded endonuclease (Yudelevich, 1971) which fragments one of the major host leucine tRNAs into two pieces 48 and 39 residues long (Sueoka and Kano-Sueoka, 1970). These two pieces remain hydrogen bonded together. Kano-

Sueoka and Sueoka (1969) have evidence that indicates that this split host tRNA can no longer participate in protein synthesis. Pinkerton et_ al. (1972) have determined the complete sequence of the T4 tRNA. Like the host tRNA, the phage tRNA is 87 nucleotides long and contains many other familiar features of the host tRNA. However, the phage tRNA-leu contains two additional modifications in the anti­ codon loop, tentatively identified as 2 -thiomethyl-6 -iso- pentenyl adenosine and an unidentified pyrimidine derivative.

This phage tRNA recognizes the codon UUA but not UUG

(Scherberg and Weiss, 1972). This phage tRNA apparently does not take the place of the cleaved host tRNA-leu, however , since the host tRNA recognizes CUG (Kan et_ al. ,

1970).

Subak-Sharpe and Hay (1965) proposed that viral tRNAs might be used to translate viral message, especially in hosts where the virus and host GC differ significantly. Presumably, in these hosts the protein synthesizing apparatus is ideally balanced for messenger with a GC representative of the host.

The base composition of T4 is 34 percent GC while that of

E. coli is 50 percent GC. T4 infection results in the pro­ duction of seven phage coded tRNAs. These are the tRNAs for leucine, proline, glycine, arginine, isoleucine, serine and glutamine (Daniel et_ al. , 1970; Weiss et_ aT. , 1968;

Daniel et^ al., 1968a, b, 1969; Scherberg and Weiss, 1970; Littauer and Daniel, 1969; McClain, 1970; Coiner et al. ,

1974). tRNA synthesis is an early gene function and Wilson and co-workers (1972) have found that all the genes trans­ cribed are closely clustered on the light strand of the phage

DNA (Scherberg et_ aJ. , 1970). Indeed, Scherberg and Weiss

(1972) have shown that phage tRNAs are more effective in polypeptide synthesis using T4 message than they are in

translating host message.

Not all phage, however, code for their own tRNAs.

Bacillus subtilis phage SP01, coliphage T7 (Weiss, 1971),

lambda (Marcaud et al_. , 1971), and ?$80 (Piecznik et al. ,

1972) do not code for their own tRNAs.

tRNA Regulation of Biosynthetic Pathways

Cognate aminoacyl tRNA synthetases are involved in repression of the biosynthetic pathways leading to the

synthesis of histidine (Brenner and Ames, 1971), lysine

(Boy et_ al^. , 1976), valine (Neidhardt, 1966), isoleucine

(Szentermai et_ al. , 1968; Jackson £t al . , 1974) and leucine

(Alexander ejt al. , 1971). In contrast, mutants with defective

(reduced affinity for the cognate amino acid) aminoacyl tRNA

synthetases for arginine (Hirshfield et^ al. , 1968), tyrosine

(Schlessinger and Nester, 1969), methionine (Gross and

Rowbury, 1969), phenylalanine (Neidhardt, 1966) and trypto­

phan (Doolittle and Yanofsky, 1968) behave like the wild type.

In order to explain the regulation of an amino acid biosynthet 24

operon by aminoacyl tRNA synthetase, it has been proposed

that 1) the aminoacyl tRNA synthetase is, itself, the apo-

repressor; 2) that both the aminoacyl tRNA and aminoacyl

adenylate are corepressors; and 3) that uncharged tRNA is

the inducer (Littauer and Inouye, 1973).

Studies by Neidhardt and co-workers have shown that the

synthesis of 10 aminoacyl tRNA synthetases for arginine,

glutamine, valine (McKeever and Neidhardt, 1976), glutamic

acid, glycine, isoleucine, leucine, lysine, phenylalanine,

and threonine (Neidhardt et al_., 1977) is correlated with

growth rate, and not with the concentration of cognate amino

acid. An exception is serine tRNA synthetase whose activity rises when exogenous serine supplies are depleted (McKeever

and Neidhardt, 1976). In a shift-up experiment (Reeh et al.,

1977) from minimal medium to glucose rich medium, the rates

of formation of five synthetases examined (arginine, glycine,

isoleucine, phenylalanine and valine) increased within 30 to

90 seconds after the shift, and the steady state level was reached within 2 to 3 minutes. This increased rate of syn­

thesis is due to an increased level of messenger RNA tran­

scription.

Ames and co-workers (Brenner and Ames, 1971) studied

the histidine operon of Salmonella typhimurium and mapped

six regions that control the operon. Ten enzymes are required for the biosynthesis of histidine. Only the hisO 25

(operator) region is linked to the operon. The other five genes are hisS (gene for histidyl tRNA synthetase), hisR

(structural gene for tRNA-his), hisU and hisW (both of which are thought to be involved in tRNA maturation) and hisT.

Cells mutant in the hisT locus contain wild-type amounts of charged and total tRNA-his, but the tRNA has an altered chromatographic mobility. This is due to undermodification of two consecutive uridine residues near the anticodon. In wild type cells, these uridines are modified to pseudouridine.

The remainder of the tRNA sequence is identical to the wild type even to the presence of a normally modified uridine (to pseudouridine) in the GTpsiC sequence (Singer et al_. , 1972;

Singer and Smith, 1972). Cells mutant in the hisT locus are derepressed for histidine biosynthetic enzymes yet have wild type levels of charged histidine tRNA (Brenner and Ames,

1972; Lewis and Ames, 1972).

Thus, the hisT mutants lack an enzyme that converts uracil to pseudouridine in tRNA. A second enzyme function is responsible for formation of pseudouridine in the GTpsiC loop. Both modifications are necessary for histidine tRNA to function in repression. In addition, other tRNAs that normally contain pseudouridine, have uridine in its place as indicated by their altered mobility on reversed phase chromatography (Cortese et_ al., 1974; Singer et al., 1972).

The hisT mutation results in partial derepression of the isoleucine-valine and leucine enzymes, furthermore Bresalier et al (1975) have shown that the hisT mutation also reduces the maximal level of expression of this pathway. Bruni et_ al. . (1977) and Lawther and Hatfield (1977) have recently identified similar pleiotropic effects in hisT mutants of

E. coli K-12 that result in lowered rates of cell growth and altered chromatographic mobilities of pseudouridine containing tRNAs. Brown et^ al_. (1977) have found that the hisT mutation does not affect tyrosine repression of 3 -deoxy-

D-arabino-heptulosonic acid-7-phosphate synthetase or tyrosine aminotransferase. Nor is there any detectable alteration in methionine repression of O-succinylhomoserine synthetase or lysine repression of beta-aspartokinase.

Indeed, Boy et^ al (1976) had shown that tRNA-lysine was not the only corepressor molecule for the lysine regulon.

Regulatory mutants (hisS, hisR, hisl? and hisW) are also derepressed for histidine biosynthesis. This derepression can be explained by the lower concentrations of charged tRNA-his in these cells (Lewis and Ames, 1972) and not with either a change in the ratio of charged to uncharged tRNA or an increase in the absolute amount of uncharged tRNA.

Mutants in the hisO locus contain wild-type levels of charged tRNA-his, yet sire derepressed as expected for mutations in the operator gene.

None of the six classes of regulatory mutants discussed above has been shown to alter any protein that serves directly as a repressor or an activator. Several lines of evidence 27

(Goldberger, 1974) suggested that the hisG enzyme ATP phospho- ribosyl transferase, is the regulatory element. However, work by Scott et^ al^. (1975) has shown that regulation of histidine enzymes occurs normally in cells carrying any deletion with both endpoints in the hisG gene. Thus, the hisG protein is not the repressor element for the histidine regulon.

The branched chain amino acid biosynthetic pathways are also regulated in part by transfer RNAs (Brenchley and

Williams, 1975; Szentermai and Horvath, 1976). Leucine biosynthesis is repressed by leucine alone; but all three branched chain amino acids, leucine, isoleucine, and valine are required for repression of the isoleucine-valine bio­ synthetic enzymes. Both the leucine biosynthetic enzymes and the transport activity for leucine are repressed by growth on leucine, but Quay and co-workers (1975) have shown that in E. coli K-12, S. typhimur ium and E. coli B/r, the transport and biosynthetic enzymes are not regulated coordinately, although their data do not rule out the possibility that the two systems may have some components in common. Indeed, they found (Quay and Oxender, 1976; Quay et al. , 1975a) that the .repression of transport involved the interaction of leucine, tRNA-leu and leucine aminoacyl tRNA synthetase. Regulation of the isoleucine-valine biosynthetic enzymes occurs at the level of transcription (Childs and Freundlich,

1975; Childs et_ al. , 1977). Using a hybridization assay with defective phage lambda CI857St68h80 dilv DNA carrying the ilv genes of E. coli K-12 Childs and co-workers (1977) demonstrated increased rates of ilv mRNA synthesis in a hisT strain as well as in tRNA synthetase mutants for iso­ leucine or leucine.

Work by Yanofsky on the tryptophan operon has revealed even more complexities in the regulation of amino acid biosynthetic pathways. The tryptophan operon of E. coli has two transcriptional control sites -- the operator and attenuator sites -- preceding the first major structural gene,of the operon (trpE). The operator site is the site of binding of the trp-aporepressor:L-tryptophan complex.

Binding of the complex to the operator site blocks the initiation of transcription (Rose et aJL. , 1973). Once transcription has initiated, it may be terminated in the attenuator site by the action of rho factor (Korn and

Yanofsky, 1976) or continue into the structural genes of the operon (Jackson and Yanofsky, 197 3; Bertrand and Yanofsky,

1976). In a mutant with an altered structural gene for tRNA-trp (trpT) the trp operon enzymes and messenger RNA levels are seven-fold higher than in the wild type (Yanofsky and Soli, 1977). These increases are due to a decrease in 29 the frequency of transcription termination at the attenuator site. Similar increases were observed in a mutant with an altered (lower Km) tryptophanyl-tRNA synthetase. Under con­ ditions which allow charging of a greater fraction of cellular tRNA-trp, a normal termination frequency is observed in both mutants. These results suggest that attenuator mediated termination is dependent on the level of charged tRNA-trp and not on the availibility of tryptophan. A trpX mutant which contains a tRNA-trp that exhibits an altered mobility on BD-cellulose columns (Joseph and Muench, 1971) exhibited increased expression of the operon. Transductional studies showed that the trpX mutation was not a class of trpT mutant.

The increased operon expression could not be reduced by aminoacylation of the tRNA-trp. Apparently, base modifications are necessary for tRNA-trp to interact at the attenuator site. The altered BD-cellulose mobility was thought to be due to a lack of modification of bases. Merodiploid studies with trpT heterozygotes could not distinguish if charged or uncharged tRNA-trp was the regulatory signal for the attenuator. The results were consistent with the idea that both charged and uncharged tRNA-trp may act to influence the attenuator.

The regulation of amino acid biosynthetic pathways is a complex phenomenon involving the participation of many com­ ponents including: aminoacyl-tRNA synthetases, rho-factor, 30 the amino acid end-products themselves, the operator, the attenuator and charged and uncharged tRNA. How all of these components fit into the regulatory system, and what undis­ covered components may also participate is a topic of current research and will be a subject of interest for some time to come.

Rationale for Studying Leucine Starvation

Leucine starvation results in a rapid termination of protein synthesis. This is a major reason why leucine

starvation has been used to block protein synthesis. I have already discussed the regulatory roles of transfer RNAs

in regulating amino acid biosynthesis and some of the changes that occur in transfer RNAs under varying physio­

logical conditions. It is possible that transfer RNAs may function as regulatory signals for other cellular systems as well.

Inspection of a table of codon assignments (Mahler and

Cordes, 1971) reveals that there are six codon assignments for leucine, for arginine and for serine. These amino acids share the distinction of being specified by more codons than any other amino acid. This suggests that these amino acids are of great importance to the functioning of the cell. This importance makes these amino acids likely candidates for regulatory signals. 31

Serine serves as a precursor in the synthesis of glycine, cysteine and methionine. Thus, a serine starvation can be considered to be a quadruple amino acid starvation. Since we are concerned with single amino acid starvations, serine starvations were not done.

Copeland and co-workers have already identified leucine and methionine as having what appears to be a unique role with regard to DNA replication.(Copeland, 1969, 1971a, b;

Copeland et_ al. , 1976). When cells are starved for leucine or methionine for two generations, nearly one half of the chromosomes do not replicate to completion. In contrast, during arginine starvation (Phillips, 1974; Copeland et al.,

1976) over 80 percent of the chromosomes replicate to com­ pletion. Consequently, leucine starvations were performed and the tRNA-leu analyzed in an attempt to detect a special regulatory function for leucine tRNA. Methionine was not chosen for this study due to its special functions as an

initiator of protein synthesis (as N-formyl methionine) and as a methyl donor for many biological reactions.

Transfer RNA: Effect of Amino Acid Starvation

Amino acid starvation results in the inhibition of bacterial RNA synthesis (Sands and Roberts, 1952). In

1961 Stent and Brenner discovered a genetic locus for the regulation of RNA synthesis. They showed that during methionine starvation, methionine requiring cells of E. coli

J-53 synthesized RNA at 10 percent of the control rate for one generation time and then stopped RNA synthesis altogether.

Under these conditions K12-W6 continued to make RNA at 66 percent of the control rate before RNA synthesis came to a halt. The continuation of RNA synthesis during methionine starvation in K1 2 -W6 can not be due to methionine, for some reason, being incapable of regulating RNA synthesis, since cells of J-53 behave normally in terras of RNA synthesis during methionine starvation. Cells of K12-W6 were referred to as "relaxed" in RNA control, whereas cells of the J-53 type were referred to as "stringent." Stent and Brenner proposed the designation RCs'tr and RCre^ (now r el + and r el -) to designate cells that were, respectively, stringent or relaxed in their control of RNA synthesis during amino acid starvation. The authors conjectured that amino acid star­ vation results in the accumulation of non-arainoacylated

(uncharged) transfer RNA. This uncharged tRNA was thought to act as a repressor of RNA synthesis in stringent cells.

Ezekiel (1964) studied the effects of chloramphenicol treatment or isoleucine starvation on the intracellular charging of E. coli tRNA. He found that within 40 minutes of combined isoleucine and valine starvation, the rate of

RNA synthesis had dropped to less than 10 percent of control

(unstarved) rates. In addition, between 80 and 90 percent of the tRNA-ile was now sensitive to periodate oxidation while only 30 percent of the tRNA-ile of control cells was periodate sensitive. Aminoacylated tRNA is protected from 33 periodate oxidation while uncharged tRNA is not. Uncharged

tRNA contains a cis-diol at the 3 1-end which is oxidized by sodium meta-periodate (sodium periodate). When tRNA is

oxidized in this fashion, it can no longer be aminoacylated.

Increasing concentrations of chloramphenicol resulted

in increasing residual rates of RNA synthesis and increased proportions of periodate insensitive tRNAs. Chloramphenicol blocks protein synthesis, the major route of tRNA de-amino-

acylation. This spares the amino acids from participating

in protein synthesis and thus from de-aminoacylating transfer

RNA. The result is a high level of charged transfer RNA.

These results supported the conclusion of Stent and Brenner

(1961) that the effect of chloramphenicol on RNA synthesis

was a consequence of its "sparing effect" on amino acids

that would otherwise be involved in protein synthesis.

Morris and DeMoss (1965) further studied the role of

aminoacyl tRNA on the regulation of RNA synthesis in E. coli.

During logarithmic growth there were characteristic and

reproducible levels of charging for the three classes of

tRNAs studied. Seventy five percent of the valine and

seventy percent of the leucine accepting tRNAs were charged

with their respective amino acids while 25 percent of the

arginine tRNAs were charged. These levels correspond,

respectively to 0.35, 0.45 and 0.26 nanomoles chargeable

RNA per mg of RNA. During leucine starvation, both the 34 arginine and the valine accepting tRNAs remained charged at their pre-starvation levels, while the periodate resistant specific acceptor activity for leucine tRNA dropped from

70 percent to 40 percent. This residual 40 percent protected tRNA-leu was considerably higher than the 10 to 20 percent level for tRNA-ile observed by Ezekiel during isoleucine starvation. Several possible explanations for this high level of tRNA-leu protection were advanced including

1) the existence of a tRNA-leu periodate protector molecule other than leucine; 2 ) the existence of an "inert" fraction of tRNA-leu; and 3) a high enough rate of protein turnover to maintain the 40 percent level observed.

A quantitative labelling experiment (Morris and DeMoss,

1965) with -^C leucine showed that the periodate protection of leucine specific tRNA could be entirely attributed to leucine itself. Methylated albumin-celite column chroma­ tography of untreated and periodate oxidized tRNA samples from growing and leucine starved cells showed identical elution patterns, indicating that the relative amounts of each leucine specific tRNA species remained the same after starvation. The high level of leucine charged tRNA-leu was shown to be due to a protein turnover level of five percent per hour. I will return to a discussion of these experiments later, but there are three points of criticism with regard to this study. First, methylated albumin celite (MAC) chroma­ tography can separate only two broad peaks of leucine 35 acceptor activity, while six isoacceptor leucine tRNAs are known, and can be resolved by other methods. It is possible that a minor peak of leucine accepting activity changes during leucine starvation, but the MAC columns could not resolve it. Secondly, the method used to starve the cells was to add a limiting amount of leucine to the medium and to allow cell growth to continue until growth was halted due to leucine limitation. This slow change in growth rate can allow any of a number of other cellular systems to be perturbed in such a way that transfer RNA charging was indirectly affected. Finally, Tockman and

Void (1977) have shown that periodate treatment of some

B. subtilis tRNAs alters their pattern of elution on

RPC-5 (reversed phase column-5) columns so that appropriate controls must be done to identify tRNAs after periodate treatment.

The most exhaustive and complete study to date of the effects of amino acid starvation on transfer RNA was that of

Yegian, Stent and Martin in 1966. These authors studied the intracellular condition of E. coli transfer RNA with respect to the degree of aminoacylation _in vivo in logarithmically growing and leucine starved cells. The strains of E. coli s*tr chosen for study were C600 (Met-, Leu-, Thi-, RC ) and

W1305 (Met-, Leu-, RC1^ 1). The actual method used for leucine starvation was not specified, although .the authors do state that starvation was for a period of time equal to 36 one division cycle. Transfer RNAs were extracted under conditions that minimized disruptions of aminoacyl tRNA ester linkages. Subsequent measurements were made of both total capacity and of acceptor capacity protected from periodate oxidation. Determinations were made for each of

sixteen of the twenty common amino acids. The four amino

acids omitted from study were asparagine, glutamine, cysteine, and tryptophan.

In both the stringent and the relaxed strains during exponential growth, most species of tRNA are periodate- protected in excess of 70 percent of the total acceptor capacity. The only anomalies were isoleucine, aspartate,

serine and tyrosine tRNA classes, whose degrees of protec­ tion are at fifty percent or less. The low degree of pro­

tection of isoleucine tRNA in exponentially growing bacteria, as well as its protection in excess of one hundred percent during leucine starvation was shown to be due to an anomaly

in the amino acid transfer reaction of tRNA-ile. The low degree of protection of tRNA-asp and tRNA-tyr reflected a

sensitivity of these tRNA molecules to periodate oxidation

at sites other than the 3’-adenosyl terminus since control

experiments with fully esterified tRNAs of these species still

showed partial periodate destruction of their amino acid

acceptor capacity. Rizzino and Ffeundlich (1975) have shown

that residual iodate (IO3 ) from periodate oxidation can

inhibit the synthetase reaction. This may help to explain 37 these lower values. In addition, during leucine starvation, the degree of protection of nearly all other classes of tRNA was even higher than in exponential growth, with the expected exception of tRNA-leu whose degree of protection fell to 13 percent for the relaxed strain and 20 percent for the stringent strain. These results are in approximate agreement with those of Ezekiel who found 15 percent periodate protected tRNA-ile during isoleucine starvation, and

Morris and DeMoss who found 40 percent periodate protected tRNA-leu during leucine starvation.

Clearly, during single amino acid starvation of E. coli the level of charging (as indicated by periodate protection) of cognate tRNA does not drop to zero. Some fraction of the tRNA is always charged. It is not clear, however, if this fraction is always the same fraction in leucine starved cells, or if it is a unique fraction -- perhaps even a single isoacceptor -- that remains charged.

In the same study, the authors analyzed the supernatant remaining after ethanol precipitation of alkali-treated tRNA.

Alkali treatment disrupts the aminoacyl tRNA ester. It was expected that the number of moles of cognate amino acid in the supernatant should correspond within experimental limits

(approximately 20 percent) to the level of periodate pro­ tection of the tRNA. This was observed for only three amino acids: phenylalanine, proline, and tyrosine. For alanine, glutamate, leucine, methionine, threonine and valine, there 33 was an excess in periodate protected acceptor capacity over

the amount of amino acid recovered. This excess was referred

to as excess protected capacity. No satisfactory explanation

of the excess protected activity was presented, except for

the case of methionine where it was shown that the excess protected activity of methionine tRNA was due to protection by N-formyl methionine.

It should be noted that the excess protector capacity

of leucine tRNA was highest for the unstarved relaxed cells

(2.07) next highest in the unstarved stringent cells (1.36)

and lowest in the leucine starved stringent cells. Leucine

starved relaxed cells showed a negative value (-0 .1 2 ) for protected capacity. That is, more amino acid was released

than was expected to account for the observed degree of periodate protection.

Yegian and Stent (1969) further investigated the excess periodate protection found only in stringent cells. The

total leucine acceptor capacity of a nucleic acid extract of

E. coli C600 (Leu-, Thr-, Thi-, RCstr) was 3.10 nanomoles

leucine per mg tRNA. After periodate oxidation of the

nucleic acid extract of a leucine starved culture, the

residual protected acceptor capacity was 0.63 nanomoles

leucine per mg tRNA. This is about 20 percent of the total

leucine acceptor capacity. When this residually protected

tRNA was subjected to alkaline hydrolysis 0.34 nanomoles

leucine per mg tRNA were released. This left 0.63-0.34 or 0.29 nanomoles of protected tRNA per mg tRNA -- about 10 percent of the total leucine accepting tRNA — insensitive to periodate. This protection is not removed by alkali treatment. Therefore, during leucine starvation, 80 percent of the leucine tRNA of strain C600 is non-acylated, 10 percent is acylated with leucine and 10 percent is protected by some substance that is neither leucine nor any of the other common amino acids. This alkali stable protector of leucine tRNA appears only during leucine starvation since during exponential growth less than 0 . 2 percent of the leucine tRNA is stably protected from periodate oxidation.

An alternative hypothesis that instead of a protector, there was some physical feature of the stably protected tRNA that made it resistant to periodate oxidation (for example, a 3 '-deoxyadenosine terminus) was tested by determining whether the stably protected fraction could retain its periodate resistance after aminoacylation with leucine, followed by mild alkaline hydrolysis. The sample was then periodate oxidized and the surviving stably protected tRNA molecules were acylated in vitro with 12C leucine. This

12C leucine aminoacylated sample was again deacylated by mild alkaline hydrolysis and the deacylated fraction oxi­ dized with periodate. Finally, this product was aminoacylated with leucine. If the stably protected tRNA was pro­ tected from periodate oxidation by a physical feature of the tRNA molecule, then that tRNA should remain stably protected throughout the above treatment and should accept the -^C leucine. If, on the other hand, the periodate stability results from the presence of a protector molecule which can be removed by the -*-2C leucine charging; then the tRNA-leu should be susceptible to the second periodate oxidation and no -^C leucine accepting capacity should survive. The results showed a leucine acceptor capacity of less than 0.01 nanomole leucine per mg tRNA, corresponding to less than one percent of the initial leucine accepting capacity of the tRNA-leu.

The 0.01 nanomole leucine acceptor activity per mg tRNA remaining showed that over 95 percent of the periodate protector capacity (initially 29 nanomoles per mg tRNA) was removed by aminoacylation. Thus, the periodate protector capacity is dissociable from the tRNA and does not result from a physical feature of the molecule.

To further study the requirements of the removal of protector from tRNA-leu, a nucleic acid extract containing a stable leucine capacity was treated with alkali, oxidized with periodate and then incubated for twelve minutes .in an aminoacylation mixture containing synthetases, but no adenosine monophosphate (AMP), pyrophosphate, adenosine triphosphate (ATP) or any added amino acids. Under these conditions, the half-life of the leucyl-tRNA-leu bond is

61 minutes. Then this enzyme treated extract was treated 41 to periodate oxidation. The extract treated in this manner could accept only 0.01 nanomoles leucine per mg tRNA while a non-treated control accepted 0.29 nanomoles leucine per mg tRNA. Thus, the protector can be removed enzymatically in the absence of AMP or pyrophosphate and therefore, can not represent the simple reversal of an aminoacylation react ion.

As part of this same study, the stable acceptor capac­ ity of the nucleic acid extract bf a leucine starved cul­ ture was measured for 16 of the 20 standard amino acids.

These measurements showed that during leucine starvation, the total stable leucine capacity was 9 percent while the stable capacity of all other amino acids tested was never greater than 1.7 percent for any other single amino acid.

To determine the species distribution of this protecting capacity, one portion of a nucleic acid fraction of a leucine starved culture was alkali treated, oxidized and then acylated with leucine. A second nucleic acid fraction was acylated with leucine without oxidation. The two portions were mixed and jointly chromatographed on a MAK column (Mandell and Hershey, 1960). The -^C-leucine activity resolved into two main peaks designated "simple peak I” and "complex, peak II." The 9 percent of the total leucine accepting capacity which represents the alkali stable, periodate 42 protected fraction was found only in peak I, showing that

during leucine starvation the protector attached only to

species I tRNA-leu.

Single amino acid starvations for histidine, threonine,

and arginine were also performed. Determinations of total, protected, and stable acceptor capacities were made. During

each of these starvations, significant residual protection

for the homologous tRNA was observed. However, with the sole exception of leucine starvation, none of the residual pro­

tected capacity was stable to mild alkaline hydrolysis.

Finally, in a comparison of stringent and relaxed cells

starved for leucine, Yegian and Stent found that the alkali

stable protector capacity was only associated with the

stringent phenotype. The authors considered three possi­

bilities for the nature of the protector: 1 ) an amino acid

derivative esterified by its carboxyl group to the terminal

adenosine, removed by a special enzyme; 2 ) a phosphate or

a phosphorylated compound such as a nucleotide, removed by

a phosphatase; or 3 ) an exchangeable terminal nucleoside

whose stepwise removal and replacement could be catalyzed by a specialized enzyme. Whatever its identity, the finding

that only species I of the several leucine tRNA species is

stable protected during leucine starvation lends support to

the idea that species I of leucine tRNA may have a regulatory

role in the metabolism of E. coli and other bacteria. 43

This is where the problem has remained. No one has

yet identified the stable leucine protector although recent

unpublished work by Holmes has confirmed the existence of a

stable leucine protector produced during leucine starvation

coli (Holmes, personal communication). However, the protector still remains unidentified.

Tockman and Void (1977) have recently published a

study on the in vivo aminoacylation of Bacillus subtilis

tRNA during logarithmic growth and in stationary phase cells

at approximately stage III of sporulation. Their method for

obtaining cells in logarithmic growth was to suspend SxlO'*'^

spores in one liter of tryptone broth. The spores had previously been suspended in sterile water and heat shocked

for 10 minutes at 80° C. Logarithmically growing cells were

harvested at an absorbance at 660 nanometers ( A ^ q ) of 1.15.

Stationary phase cells were harvested at an A ^ q of 5.70.

Due to the methods used to obtain logarithmically growing

cells and due to the fact that the medium was undefined

their results must be interpreted with some caution.

Nevertheless, Tockman and Void found a level of leucine

charging of 58 percent in logarithmically growing cells and

of 62 percent in stationary phase cells. Individual tRNA profiles were presented for the methionine, lysine, and

tyrosine tRNAs of stationary phase cells, with and without periodate treatment. Their results showed that cognate tRNA species of phenylalanine, methionine, glutamic acid, leucine and proline showed normal chromatographic behavior after periodate oxidation since oxidized and unoxidized material gave the same elution profile. However, lysyl-, tyrosyl-, and tryptophanyl tRNAs showed altered elution patterns after periodate treatment. Thus, appropriate controls must be done when examining these species of tRNA after periodate treatment. No interpretation was made in terms of the relative charging levels of any isoacceptor between logarithmic and stationary phase, although their results indicate a relative increase in methionine acceptor capacity for RPC-5 peak I in stationary phase after periodate treatment.

As stated in the Introduction, I have undertaken a study to determine: 1) the overall levels of leucine tRNA aminoacylation during balanced growth and during leucine starvation and 2 ) furthermore to examine the relative changes in charging levels for each of the separable leucine iso­ acceptors by separating these charged isoacceptors on Sepharose

4B columns. The results of this study showed differences in balanced growth aminoacylation levels of the two strains of Bacillus subtilis studied. In contrast, during leucine starvation, both strains exhibited equivalent lower levels of leucine tRNA aminoacylation. Furthermore, Sepharose 4B chromatography resolved the total leucine accepting activity into three peaks, each of which decreased proportionately with the overall decrease in tRNA aminoacylation. MATERIALS AND METHODS

Bacterial Strains

Strains of Bacillus subtilis used in this study, their genotype and source are listed in Table 1. A map showing the relative position of genetic markers on the chromosome of B. subtilis is shown in Figure 2.

Media

Liquid minimal medium (Anagnostopoulos and Spizizen, 1961) consisted of minimal salts (0 . 6 percent, weight:volume, KH2PO4 ;

1.4 percent K 0HPO^; 0.1 percent sodium citrate*H2 0 ; 0.2 percent ammonium sulfate and 0 .1 percent magnesium sulfate added aseptically after autoclaving) and 0.5 percent glucose sup­ plemented with 50. micrograms per ml of each required amino acid. Minimal medium plates consisted of minimal salts plus glucose, amino acids, and 1.5 percent agar supplemented with

L-glutamic acid and L-asparagine at 50 nicrograras per ml.

Dilutions for plate counts were carried out in diluent con­ sisting of minimal salts. Trypticase Soy Broth (TSB: BBL,

Cockeysville, MD) was supplemented with 50 micrograms per ml adenine and thymine.

45 Table 1.

Gene symbols indicate mutation in cistrons leading t the indicated requirement as follows: git, glutaraic acid his, histidine; leu, leucine; pur, adenine or guanine; thr, threonine; and trp, tryptophan. Table 1. Strains of Bacillus subtilis

Other Strain_____ Designation______Genotype______Source

168

BC29 168-2 trp-2, leu-2 J. Pene

W23

BCW503 SB12 thr, his, leu E. Nester 48

Figure 2.

Representation of a genetic map for Bacillus subtilis showing the relative distance and order of replication for markers pertinent to this study. Adapted from J. Lepesant-

Kejzlarova, J. -A. Lepesant, J. Walle, A. Billault, and

R. Dedonder. 1975. Revision of the linkage map of

Bacillus subtilis 168: Indications for circularity of the chromosome. J. Bacteriol. 121: 823-834. FIGURE 2.

ORIGIN TERMINUS

purA16 hisA leu-8 trp gltA 50

Chemicals

Buffer saturated phenol was prepared from Matheson, Coleman and Bell reagent grade phenol (loose crystals) without prior distillation. All other chemicals were reagent grade.

Column Materials

Sepharose 4B was obtained from Pharmacia. DEAE-Cellulose

(Cellex D) was obtained from Bio-Rad Laboratories, Richmond

California.

Radioisotopes

L-leucine (4,5-%) with a specific activity of 62 Curies per millimole was obtained from Schwarz/Mann.

Enzymes

Deoxyribonuclease (DNase) and lysozyme (hen egg white) were obtained from Calbiochem, La Jolla, Calif.

Preparation of Aminoacyl tRNA Ligase (tRNA Synthetase)

Two 500 ml flasks containig 100 ml of trypticase soy broth (TSB) supplemented with adenine and thymine (50 micro­ grams per ml, final concentration) were inoculated from a single colony and incubated overnight at 37°C with shaking.

The following morning, the cultures were poured into two liter flasks each containing one liter of pre-warined TSB. The cultures were further incubated with shaking for three to four hours and iced. All subsequent operations were carried out at 4°C.

The cells were pelleted out by centrifuging at 6000 rpra

(5875xg) in a GSA rotor. The pellets were pooled and washed 51 once by resuspending in 20 ml of synthetase buffer A (SA buffer:

0.1 M Tris (pH 8.0 at 25°C); 0.1 M MgCl2 ‘6H 2 0 ; and 10 percent glycerol),and centrifuging once more. The supernatant was discarded and the cell wet weight was determined. Generally this procedure yielded between 7 and 10 grams of cells.

The pellet was then resuspended in SA buffer (3 ml of SA buffer per gram of cells. This suspension was mixed with an equal volume of glass beads (0.10-0.11 mm diameter Glasperlen;

VWR Scientific #34007) and sonicated with 4 separate 30 second bursts in a Bronwill cell homogenizer. The glass beads were separated from the cell sap by passing the mixture through a syringe plugged with glass wool. The cell sap was then cen­ trifuged in an SW50L rotor (Beckmann Instruments, Palo Alto,

Calif.) at 33,000 rpm (198,000xg) for two hours. The clear supernatant was decanted to a level even with the uppermost portion of the pellet, pooled, mixed with two ml of glycerol and loaded onto a 24x2 cm DEAE-cellulose column equilibrated with synthetase buffer B (SB buffer: 0.02 M potassium phos­ phate buffer, pH 7.5; 0.02 M 2-mercaptoethanol; 0.001 M MgC^J and 10 percent glycerol). The column was previously washed with 250-300 ml of SB buffer. The synthetases were then eluted with synthetase buffer C (SC buffer: 0.25 M potassium phosphate buffer; 0.02 M 2-mercaptoethanol; 0.001 M MgCl2 and 10 percent glycerol). Fractions (150 drops) were collected, and the absorbance of each fraction at 280 nanometers was determined. 52

The fractions with maximal A280 absorbance were then pooled, made 50 percent in glycerol, divided into 1 ml aliquots and frozen in a liquid nitrogen refrigerator (-195°C).

Synthetase could be stored under these conditions with no decrease in activity for six months. Synthetase isolated by this method contained no detectable DNA or RNA.

Starvation Regimen

A single colony was picked and inoculated into 10 ml of liquid minimal medium and incubated overnight aerobically with shaking (250 revolutions per minute) in a Controlled

Environmental Shaker (New Brunswick Scientific Co., New

Brunswick, N. J.) at 37°C. During the next day, the culture was diluted to maintain the cell population in balanced growth at about 10^ cells per ml. An appropriate dilution of this same culture was used to inoculate one liter of fresh minimal medium in a 2 liter Erlenmyer flask so that after 16-17 hours 7 of incubation the cell population would be 1-5x10 colony forming units per ml. The growth of this culture was monitored at 30 minute intervals in a Klett-Summerson colorimeter

(blue filter, number 42). When the optical density of the culture approached 20 net Klett units (gross Klett minus Klett absorbance of the vessel) equivalent to 3-5x10 cells per ml;

500 ml of the cultune was poured into a graduated cylinder packed in ice, containig 5 ml of 1 M sodium azide and RNA was isolated as described in the following section. 53

The remaining 500 ml were filtered through a 90 mm mem­ brane filter (Type B-6 ; Schleicher and Schuell Co., Keene, N.H.) and washed three times with 1 00 ml each of pre-warmed minimal medium containing all growth requirements except leucine.

The cells were resuspended in an equal volume of pre-warmed medium lacking only the amino acid under study. Ten ml of the starved culture was removed and added to a flask containing

100 micrograms per ml (final concentration) of leucine. This culture served as a control to monitor the effects, if any, of filtration on cell growth. The entire procedure of filtration and resuspension averaged less than four minutes. The optical density (Klett) of both cultures was measured and plotted.

A 0.2 ml sample was taken from the starved culture immediately after the initiation of starvation, diluted in diluent and plated on trypticase soy agar (TSA) to determine the number of colony forming units per ml, which was equal to the viable number. A similar 0.2 ml sample was taken from the starved culture after a time equivalent to 2 generations of growth of the control culture to determine the change; if any, of viable number during amino acid starvation. At times equivalent to 0, 1.5 and 2 generations of growth of the control culture, two (each) 5 . 0 ml samples were removed from the starved culture, rapidly frozen in a dry ice-ethanol bath and stored at -195°C.

These samples were used subsequently to determine DNA concen­ trations . 54

After a period of time equivalent to two generations of growth of the control culture, the contents of the flask

(400-500 ml) were poured into a graduated cylinder packed in ice containing 5 ml of 1 M sodium azide and RNA was isolated as described in the following section.

Preparation of Transfer RNA

Cells were harvested at an optical density of 20-30 Klett units. 400 or 500 ml of cells were made 0.01 M in sodium azide and iced by pouring into a graduated cylinder packed in ice. The samples were centrifuged at 4°C, 6000 rpm (5875xg) in a Sorvall GSA rotor and resuspended in 3 ml of tRNA prepa­ ration buffer A (TPA: 0.005 M MgCl2 ; 0.06 M KC1; 0.05 M sodium acetate; final pH of 5.5) and transferred to a baked

30 ml COREX tube. This tube was baked to remove RNase activity.

Lysozyme (Calbiochem, La Jolla, Calif.) final concentration of 300 micrograras per ml, and DNase (Calbiochem) final con­ centration 10 micrograms per ml, were added and the preparation was frozen and stored at -195°C until isolation could be completed.

The isolation was completed by thawing the sample, freezing once more and thawing again. Sodium dodecyl sulfate

(SDS) was added to a final concentration of 0.1 percent. The suspension was extracted with an equal volume of TPA saturated phenol for 5 minutes at room temperature. The phases were separated by centrifugation at 9,500 rpm (10,886xg) in a

Sorvall SS 34 rotor for 10 minutes at 4°C. The upper (aqueous) 55

layer was decanted to a baked 30 ml COREX tube, mixed with 0.2

volumes of 20 percent (w/v) sodium acetate, pH 5.2 and 2

volumes of cold 95 percent ethanol, and allowed to stand at

-20°C for at least 30 minutes. The precipitate was removed

by centrifugation at 9,500 rpm (10,886xg) in a Sorvall SS34 rotor for 10 minutes at 4°C and resuspended in 2 ml of 0,1 M potassium phosphate buffer (pH 6.0) and stored at -195°C.

This preparation was stable and could be kept frozen for three months.

Samples isolated in this way generally contained 500 to

1000 raicrograms of RNA per ml as determined by the orcinol test

using ribose standards and less than 1 00 micrograms per ml of

DNA as determined by the diphenylamine test using calf thymus

DNA (Calbiochem, La Jolla, Calif.) and less than 100 micrograms per ml of protein as determined by the method of Lowry et_ al.

(1953) using bovine serum albumin (Nutritional Biochemical

Corporation, Celveland, Ohio) standards.

Determination of Amino Acid Acceptor Capacity

The charging buffer consisted of 0.1 M potassium phosphate buffer (pH 6.0) containing 5mM MgS04 , 2mM 2-Mercaptoethanol and

500 micromolar adenosine-5'-triphosphate (ATP). For a single

50 microliter sample of RNA (containing between 600 and 1000 micrograms of RNA per ml) the complete charging mixture con­

sisted of 0.5 ml of charging buffer plus 0.15 ml of undiluted

synthetase (800-1000 micrograms protein per ml) and one micro-’

liter of undiluted %-leucine. 56

Saturation curves were run (see Results) to determine

the concentration of the amino acid and the dilution of the

enzyme that would allow the reaction to reach completion in

fifteen minutes. The reaction was stopped by adding (to each 0.5ml

sample) 2 ml of 10 percent trichloroacetic acid (TCA) plus

0.1 M unlabelled leucine and chilling for a minimum of 30

minutes at 4°C. The precipitate was collected by filtration

on 2.4 cm GF/A glass fiber filters (Whatman, Ltd., England)

washed 3 times with 2 ml each time of 5 percent iced TCA,

dried and placed into a 17x60 mm, 2 dram vial containing 5 ml

of toluene scintillation cocktail (3 grams of 2,5-diphenyl-

oxazole in one liter of toluene). The vials were counted in

a Packard or Nuclear Chicago scintillation counter.

Periodate Treatment of tRNA

Treatment with sodium periodate (NaI0 4 ) will oxidize

the adjacent 2', 3'-diol group of the terminal ribose in

uncharged tRNA to form adjacent aldehydes with concomitant

cleavage of the ribose ring (Berg et al_. , 1961). Aminoacylated

tRNA contains an amino acid linked as an ester to one of the

free hydroxyls. Aminoacylated tRNA is, therefore, resistant

to periodate oxidation.

The following procedure was used to treat the RNA samples

to determine the percentage of aminoacylated tRNA.

Duplicate samples of RNA were pipetted into 13x100 mm

Pyrex test tubes. To inactivate uncharged tRNA an equal volume 57 of 10 mM sodium periodate was added to one RNA sample and incubated at 37° C for 15 minutes in the dark. The control sample received an equal volume of water. A volume of 35 mM sodium hydrogen sulfite (NaHSO^) equal to the volume of sodium periodate used was then added to each tube and thereaction was allowed to proceed for 10 minutes at 37° C inthe dark.

Sodium hydrogen sulfite was added to remove periodate and iodate

(a by-product of the oxidation of ribose) from the reaction mixture according to the following reaction scheme (Sienko and

Plane, 1966):

2 1 O4 + 2HS0" — > 2 IO3 + 2So|" + 2H+

2 I0 § + 5IISO3 — > 12 + SSO^" + 3H+ + H20

Net reaction:

210" + 7 HSO3 — > 12 + 7SoJ" + 5H+ + h20

Two volumes of cold 95 percent ethanol plus 0.2 volumes of

20 percent sodium acetate (pH 5) were added to each sample and the samples were iced five minutes in a dry ice-ethanol bath.

Samples were then centrifuged at 4° C and 5,000 rpm (3079xg) in a Sorvall 3424 rotor for 15 minutes. The supernatant was discarded. 50 microliters of sodium hydrogen sulfite and 0.5 ml of 0.05 M Tris(hydroxymethyl)aminomethane; pH 8.01 at 37° C were added to each sample and allowed to stand for 60 minutes at 37° C in the dark to strip charged tRNA (von Ehrenstein and

Lippmann, 1961). Then 2 ml of cold ethanol and 0.2 ml of 58

20 percent sodium acetate were added to each sample. They were then iced in a dry ice-ethanol bath for five minutes and centrifuged at 5,000 rpm (3079xg) and 4°C for 15 minutes in a Sorvall SM-24 rotor. The supernatant was discarded. The

RNA in the pellet was then aminoacylated as described above.

Sepharose 43 Chromatography

Individual isoaccepting tRNAs were resolved by Sepharose

4B column chromatography after the method of Holmes et al.

(1975). To prepare the column, Sepharose 4B was diluted with water and poured into a 1.5x40 cm glass column. The Sepharose was then equilibrated with tRNA preparation buffer C (TPC:

0.01 M magnesium acetate*4 H2 O; 6 mM 2 -mercaptoethanol; 10mM

MgCl2 ‘6H2 0 ; and 1 mM ethylenediaminetetra-acetate (EDTA); final pH of 4.5 at room temperature) by passing three column volumes of TPC through the column. The column was then washed with three column volumes of TPC containing 2M ammonium sulfate (TPC+2) and stored at 4°C. Periodate treated aminoacylated samples in TPC+2 containing glucose

(to aid in loading the column) were applied to the Sepharose

4B column and eluted at 4°C with a gradient of 175 ml of

TPC+2 in the mixing chamber and 500 ml of TPC in the reservoir, at a flow rate of 20 ml per hour. 150 drop fractions were collected and refractive index and absorbance at 260 nanometers were measured. Radioactivity was determined by pipetting 0.1 ml of the fraction into a 17x60 mm, 2 dram vial containing 5 ml of aquesous scintillation cocktail 59 consisting of 6 .grams of 2,5-diphenyloxazole; 0.4 gm of

£-2-(5-phenyloxazolys) benzene; 200 ml of Bio-Solv (Beckmann

Instruments, Inc., Fullerton, Calif.) in one liter (final volume) of toluene.

Chemical Determinations of DNA

DNA was determined by a modified Schmidt and Thannhauser

(1945) procedure. Thawed 5.0 ml samples were centrifuged at

4°C and 5000 rpm (3079xg) in a Sorvall SM24 rotor for 20 minutes. The supernatant was discarded and the pellet was re-suspended in 2 ml of 10 percent trichloroacetic acid

(TCA: Fisher Scientific Co., Pittsburg, Penn.) and iced a minimum of thirty minutes. Samples were centrifuged once again, washed with an equal volume of 10 percent TCA and then re-suspended in 0.2 ml of 1 M K0H. The tubes were sealed with Parafilm (American Can Company, Neenah, Wise.) and incubated 15-18 hours at 37°C. Each sample was then pre­ cipitated with 0.8 ml of a stock acid mixture (5.5 ml of

1 M HC1; 12.3 ml of 10 percent TCA; and 2.2 ml of H2 O) and iced a minimum of one hour. The pellets and supernatant were separated by centrifugation for 30 minutes at 4°C.

Acetone (0.5 ml) was added to each sample and the samples were dried at room temperature _in vacuo.

DNA determinations were made on pellets by adding 0.1 ml of 10 percent (w/v) perchloric acid and 0.1 ml of freshly prepared diphenylamine (Fisher Scientific Co., Pittsburg, PA) 60 in glacial acetic acid containing acetaldehyde (80 micrograms per ml). Tubes were sealed with Parafilro and allowed to stand for 12 to 16 hours at room temperature in the dark.

Absorbance was measured at 595 nanometers and 700 nanometers.

The absorbance at 700 nanometers was subtracted from that at 595 nanometers to correct for light scatter due to parti­ cles in suspension. Standard curves were obtained in all cases using Calf Thymus DNA (Calbiochem, La Jolla, Calif.).

Chemical Determinations of RNA

0 . 2 ml of the sample to be analyzed were mixed with 0 .2 ml of orcinol reagent (60 rag orcinol; 50 mg FeC^'bl^O; and 6 ml stock HC1), boiled in water for 15 minutes and the absorbance at 660 nanometers was determined. Ribose stan­ dards were included in all determinations.

Chemical Determinations of protein

Protein concentration was determined by a modification of the method of Lowry et_ al. (1953). Samples (0.1 ml) and standards were pipetted into 13x100 mm Pyrex tubes.

1 ml of 10 percent TCA was added to each tube and the tubes

were iced a minimum of 5 minutes. Samples were centrifuged at 7,000 rpm for 10 minutes at 4°C in a Sorvall SM-24 rotor. The supernatant was discarded and 0.5 ml of cold

10 percent TCA was added. The samples were centrifuged again and the supernatant was discarded. 0.1 ml of 1 M

NaOH was added to each tube. Tubes were vortexed and 61 incubated at 37°C for 30 minutes. 1 ml of reagent C (0.1 ml of 2 percent sodium potassium tartarate*4H2 0 ; 0.1 ml of 1 percent CuS04 *5H2 0; 5 ml of 4 percent sodium carbonate and 5 ml of water) was added, vortexed and incubated exactly 10 minutes at 37°C. 0.1 ml reagent D (1:2 dilution of commercial Folin phenol reagent; Fisher Scientific,

Cleveland, Ohio) was added to each tube, vortexed and incu­ bated exactly 10 minutes at 37®C. Samples were then placed in a water bath at room temperature for 10 minutes and the absorbance at 750 nanometers was determined. Protein stan­ dards contained bovine serum albumin (Nutritional Biochemi­ cal Corp., Cleveland, Ohio).

Sucrose Density Gradient Analysis

To determine the amount of tRNA relative to other RNA in the RNA preparation, 0.1 ml of the extracted RNA was layered on 12 ml of a linear (6 to 21 percent) sucrose gradient in 0.01 M Tris; 1 mM EDTA and 50 mM NaCl; final pH 7.0. The 9/16 by 3% inch cellulose nitrate tubes

(Beckmann Instruments, Fullerton, Calif.) were centrifuged at 25,000 rpm (110,000xg) in a Beckmann SW41 rotor at 4°C for 20 hours. After centrifugation, the bottom of the tube was punctured and the contents were pumped through an ISC0 model 222 UV-analyzer (ISCO, Lincoln, Nebraska). The absorbance at 254 nanometers was continuously monitored. The area under the absorbancy tracing was determined for the tRNA. region and for the total RNA region, by the method of weighing paper cut-outs. The ratio of the two areas was determined. The percentage of tRNA equals

area of the tRNA region_____ area of the total RNA region RESULTS

Growth Kinetics and Viability

Cells of a 168 strain of Bacillus subtilis (BC29) and a

W23 strain (BCW503) were starved for leucine. Previous work

(Thomas and Copeland, 1973) has shown that both these strains are stringent for RNA control, that is, during leucine star­ vation, both strains showed increases in RNA or less than

10 percent.

Samples were taken at 1.5 and at 2 generation time equivalents after initiating starvation. The ratios of later samples to those at the start of leucine starvation were com­ pared for number of colony forming units, Klett readings and

DNA determinations. Comparisons of the 1.5 and 2 ratios showed that DNA synthesis had stopped by the earlier time.

Immediately after starvation, a 10 ml aliquot was removed and supplemented with leucine. This 10 ml aliquot served as a control to demonstrate that filtration produced no appreciable effect on growth rate. Starved cells of

B. subtilis re-supplemented with leucine resumed their pre­ filtration rate of growth almost immediately. When cells are deprived of leucine, there is a rapid cessation of growth

63 64 generally levelling off in less than sixty minutes with an average increase of less than fifteen percent in Klett

(Figure 3, Table 2 ). n At the time of starvation, there were 3-5 x 10 colony forming units per ml. During leucine starvation, cells of

BC29 increased slightly more in Klett than in viable number, while cells of BCW503 starved for leucine increased in viable number slightly more than they increased in Klett.

DNA Levels

The changes in total cell DNA after two generation times of leucine starvation are summarized in Table 2. The amount of DNA made during starvation is a characteristic unique to the strain being starved and the amino acid for which the strain is being starved (Copeland, et_ al. , 1976). Thus, leucine starvation results in an 18 percent increase in DNA for BC29 while leucine starvation of BCW503 results in a 26 percent increase in DNA. These results are in agreement with published observations (Copeland et_ al_. , 1976).

Preparation of tRNAs: Retention of Aminoacyl-tRNA Esters

Transfer RNA was isolated at low pH in order to maintain the integrity of the aminoacyl-tRNA ester. To determine the efficiency of the isolation scheme in maintaining aminoacyl- tRNA bonds, a sample of tRNA was fully aminoacylated with

H-leucine by the standard assay procedure for amino acid incorporation and re-isolated using the same scheme as used to isolate transfer RNA. Results are presented in Table 3. 65

Figure 3.

Growth kinetics for BC29 and BCW503 when starved for the amino acid leucine. 66

BCW503

+LEUCINE

30 -LEUCINE 25

20

O.D. (KLETT)

BC29

30 +LEUCINE

25

-LEUCINE 20

0 60 120 180

TIME (minutes) FIGURE 3 67

Table 2.

Effects of leucine starvation on cell titer, total DNA and optical density (IClett). Values are the ratios of determinations made after starvation for the equivalent of two generation times to determinations made at the initiation of starvation. Values in parentheses are standard errors. 68

Table 2. Effects of Amino Acid Starvations

Strain Titer DNA Klett

BC29 0. 92 1.08 0. 96 1. 05 0. 90 1.17 0.89 1.02 1.15 1. 27 1 . 00 1.23 1. 39 Mean: 0.96 (.02) 1.18 (.02) 1.14 (.09)

BCW503 1.19 1.33 1.10 1.27 — 1.29 1.03 1.27 0.94 1.20 — 1.05 1.12 1.17 1.07 Mean: 1.16 (.04) 1.26 (.07) 1.09 (.06) Table 3. RNA concentration and tritium activity before and after isolation3

RNA Counts Concentration per minute

Initial 1.84 ug/ml 5.3 x 10^

Final 0.52 ug/ml 4.4 x 10^

Percent Recovery 28% 83%

aAn RNA sample from BC2 9 in logarithmic growth was aminoacylated (Initial) and then re-isolated (Final) using the methods outlined in the text, in order to determine the efficiency of the isolation scheme. These results show that during re-isolation, only 17 percent of the initial tritiated leucine activity was lost. This second passage through the isolation scheme resulted in a loss of 72 percent of the RNA, indicating that the tRNA fraction as isolated contains RNA-like material that does not bind radioactive leucine. Much of this material is removed by a second purification cycle and is probably ribosomal RNA (see Sucrose Gradients). The purification scheme results in a 3 -fold purification of the leucine accepting activity (83% *- 28%). Since I am primarily concerned with relative changes in levels of aminoacylation and since all RNAs were extracted using this isolation scheme, there is no reason to doubt that this method is satisfactory for this purpose.

This experimental scheme does have some'limitations.

Later in this section, I will show that the RNA isolated from amino acid starved cells is aminoacylated to a lower degree than the RNA isolated from cells in balanced growth.

This lower level of aminoacylation seen in leucine starved cells may be real. Alternatively, amino acid starvation might affect aminoacylated tRNAs in some way so that they are more prone to de-aminoacylation during the isolation procedure. The experiment to check the isolation scheme does not distinguish between these two possibilities.

However, the lower aminoacylation level seen in amino acid starved cells does indicate that there is some difference 71 between balanced growth and leucine starved tRNA. The observations of other workers (see Literature Review) and the generally accepted reliability of the methods used to extract the tRNA in its aminoacylated form favor the explanation that leucine tRNAs are aminoacylated at lower levels during leucine starvation than in balanced growth.

Sucrose Gradients

The previous section showed that the RNA as isolated contains a substantial amount of non-leucine binding RNA.

Sucrose gradient centrifugation was used to determine what portion of the total RNA preparation was transfer RNA and to determine to what extent RNA degradation by RNase had occurred. Representative RNA preparations isolated both from growing and leucine starved EC29 and BCW503 were subjected to sucrose density gradient analysis as described in Materials and Methods. Results are presented in Figures 4, 5, 6 , and 7.

The relative areas under each curve were determined by the method of weighing paper cut-outs of the graph. These data are summarized in tabular form in Table 4. These results show that the relative amount of tRNA isolated varies from a low of 18 percent (BCW503 in balanced growth, Figure 6 ) to a high of 68 percent (3C29 leucine starved, Figure 5).

In addition, these gradients show that, while there is some breakdown of RNA, the tRNA peak is still evident. Thus, there is not extensive RNase breakdown of the RNA during isolation. 72

Figure 4.

Sucrose gradient analysis (A.2 5 4 ) of RNA isolated from

BC29 in balanced growth, p indicates direction of increasing sucrose concentration. ABSORBANCE (254 run) BOTTOM 23S I6S 4-LEUCINE BC29 4S TOP IUE 4 FIGURE 73 74

Figure 5.

Sucrose gradient analysis (A2 5 4 ) of RNA isolated from

BCZ9 starved for leucine for two generation time equivalents. p indicates direction of increasing sucrose concentration. ABSORBANCE (254nm ) BOTTOM 23S I6S -LEUCINE BC29 4S IUE 5 FIGURE 75 TOP 76

Figure 6 .

Sucrose gradient analysis (A2 5 4 ^ isolated from

BCW503 in balanced growth, p indicates direction of increasing sucrose concentration. ABSORBANCE (2 5 4 nm) BOTTOM 23S I6S LEUCINE + BCW503 4S

TOP IUE 6 FIGURE 77 78

Figure 7.

Sucrose gradient analysis (A2 5 4 ) of RNA isolated from

BCW503 starved for leucine for two generation time equi­ valents. (O indicates direction of increasing sucrose concentration. ABSORBANCE (254nm) OTMTOP BOTTOM 23S I6S -LEUCINE BCW503 4S 80

Table 4. Percentage of 4S RNA relative to total RNA

Strain_____ Condition_____ % 4SRNA

BC29 +leucine 25

BC29 -leucine 68

BCW503 +leucine 18

BCW503 -leucine 32 Thomas and Copeland (1973) have observed that during leucine starvation BCW503 synthesizes more 4jB RNA relative to 16!B and 23_S species than does BC200 (a 168 strain).

However, both strains do maintain some 4S RNA synthesis during leucine starvation. In my experiments, both leucine starved 3CW503 and BC29 show increases in the 4S peak relative to total RNA. This is in keeping with the observation of Thomas and Copeland. However, my experiment does not show whether the increase in 4j> RNA is due to continued synthesis of 4_S RNA or whether it is due to extensive breakdown of 16S and 23_S RNA. What this experi­ ment does show is that unless the relative amount of tRNA is determined for a given sample, the leucine accepting activity per mg RNA is not a quantitative term. It can vary over a three-fold range depending on what percentage of the RNA is tRNA.

Determination of Acceptor Activity: Base Hydrolysis

Prior to aminoacylation, the transfer RNA was stripped of nascent amino acid by incubation at 37°C in Tris buffer at pH 8.0. To determine the necessary incubation time, an

RNA sample was fully aminoacylated with tritiated leucine and incubated at 37°C in Tris. Samples were removed at intervals and added to 10 percent TCA containing 0.1M unlabelled leucine. They were then iced, filtered onto 82

GF/A glass fiber filters (Whatman Ltd., England) and washed with 5 percent TCA. The results of this experiment are presented in Figure 8 .

One hour of incubation at pH 8 was sufficient to reduce the TCA precipitable activity to 6 percent of the initial level. Further incubations resulted in only slight decreases in TCA precipitable activity. Therefore, prior to amino­ acylation all tRNAs were stripped by incubation at 37°C and pH 8.0 for one hour.

Morris (1966) found that under certain conditions, the charging reaction could displace unlabelled amino acids present on tRNAs with labelled amino acids present in the charging mixture, presumably through an equilibrium reaction.

I have duplicated this finding (Figure 9). The displacement reaction would make high pH de-aminoacylation unnecessary.

However, since high pH deaminoacylation is a standard part of any currently used aminoacylation scheme (Tockman and

Void, 1977) it was used prior to aminoacylation in all experiments used to determine ir2 vivo aminoacylation levels and profiles.

Kinetics of Leucine Incorporation

The optimum charging time needed to effect complete

aminoacylation was determined by adding 1 ml of stripped or unstripped RNA to 3.2 ml of the complete charging mixture and incubating at 37°C. At intervals, samples were removed 83

Figure 8 .

Kinetics of base hydrolysis of aminoacyl tRNA. A fully aminoacylated sample was incubated at 37°C in Tris

at pH 8.0. One hundred microliter samples were removed at times indicated and added to 2 ml of 10 percent TCA

containing 0. 1M unlabelled leucine, iced and then filtered onto GF/A glass fiber filters. The samples were washed

three times with two ml (each wash) of iced 5 percent TCA,

dried and counted in non-aaueous cocktail as detailed in the Materials and Methods. I ! 8 -

r H

4 -

m 00 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 TIME (minutes) AT pH 8.0 85 and added to cold TCA containing unlabelled leucine.

Samples were iced and the precipitates collected on GF/A filters. The results sure presented in Figure 9.

Aminoacylation was complete within 10 minutes of

incubation. Further incubations for 20 and 30 minutes

showed no significant increases in leucine accepting activity. Consequently aminoacylations were carried out for 15 minutes at 37°C in the standard assay mixture.

RNase Control

Since it is possible that substances other than RNA

might bind leucine in some non-specific way, an experiment

was performed to determine the effects of RNase on the

aminoacylation reaction. Fifty microliter RNA samples were

incubated in RNase (500 micrograms per ml final concentration)

for 15 minutes and 30 minutes at 37°C.. A control, sample was incubated for 30 minutes at 37°C in the absence of RNase.

These samples were then aminoacylated as described. The results of this experiment are presented in Table 5.

This experiment shows that 15 minutes of incubation

in the presence of RNase was sufficient to reduce the

leucine accepting capacity of an RNA preparation to 8 percent of the control level. Thus, the leucine accepting

activity of the RNA preparations is sensitive to RNase. 86

F i g u r e 9.

The kinetics of leucine incorporation into untreated and base-hydrolyzed tRNA. One sample (pH 10) was adjusted to pH 10 by the addition of solid Na2C0 3 ant^ incubated for

30 minutes at 37°C. The sample was then neutralized by the addition of 1.0 N HC1. A 1 ml sample of RNA was added to 3.2 ml of the complete charging mixture and incubated at 37°C. At designated intervals, 0.5 ml samples were removed and added to 2 ml of iced 10 percent TCA containing unlabelled leucine at an initial concentration of 1 0 “^M.

Samples were iced 30 minutes and the precipitates were collected on GF/A glass fiber filters. The samples were washed three times with 2 ml (each wash) of iced 5 percent

TCA. The filters were dried and counted in five ml of non-aqueous cocktail. Both samples contained a final concentration of 150 micrograms per ml ribose. 8 -

UNTREATED cn o pH 10 r“ l x

g u 4 —

M u D 3 Du CO

c 12 14 16 18 20 22 2426 28 30 X) CHARGING TIME (minutes) 88

Table 5. Sensitivity to RNase. 50 microliter samples were treated with RNase as described in the text and then aminoacylated with leucine in the standard assay mixture. "Blank" is a control and contains no RNA.

Sample cpm Percent

Control 10,962 1 0 0

15 min. RNase 851 00

30 min. RNase 806 7.3

Blank (no RNA) 264 2 89

Periodate Treatment

Sodiun periodate (SxlO-3 M) is used to inactivate non-aminoacylated tRNA (Eerg et_ al_. , 1961). Recently however, Rizzino and Freundlich (1975) demonstrated that iodate (a reduction product of periodate) can inhibit acylation of tRNA, presumably by affecting the charging reaction itself. To avoid this interference, some workers

(Tockman and Void, 1977) have added excess ethylene glycol to periodate treated RNA to reduce the periodate still present to iodate. The RNA preparations are then dialyzed overnight at 4°C. Since this dialysis exposes the RNA to

RNase attack for a considerable period of time, an attempt was made to find an alternative method to remove the inter­ fering anions.

Sodium hydrogen sulfite (sodium bisulfite) is used industrially to remove sodium iodate from Chile saltpeter

(Sienko and Plane, 1966). The principle reaction is:

5HSO3 + 2105 > I2 + 5S0|" + 3H+ + H 20.

Sodium hydrogen sulfite could also be expected to react with periodate to produce iodate by the following reaction scheme: 2 " + 2HSO3 + 2I04 — 2 IO3 + 2S04 + 2H .

The net reaction is:

210“ + 7HS0" > I2 + 7SO3' + 5H+ + H.,0.

To test this reaction scheme, the following experiments were done. To determine the total amount of chargeable 90

RNA in a sample, 50 microliters of water was added to a 50 microliter sample of unstripped RNA. The 50 microliter additions of water added here and in other experiments of this series are added to compensate for the diluting effects 50 microliter additions of periodate, sodium hydrogen sulfite and iodate have on the charging reaction.

This sample was incubated at 37°C for 15 minutes. Sub­ sequently, 50 microliters of water was added and the mixture was incubated for 10 minutes further. Then 0.5 ml of com­ plete charging mix was added to the mixture and aminoacylation carried out as described in Materials and Methods. The amount of tritiated leucine incorporated represents the total amount of chargeable RNA in the RNA'preparat ion.

This sample is labelled "Control" in Table 6 .

To determine the amount of aminoacylated tRNA present in this preparation, sodium periodate was added to RNA and allowed to react. Subsequently, sodium hydrogen sulfite was added to the mixture and allowed to react. The mixture was then aminoacylated with charging mix as described.

The periodate should inactivate non-aminoacylated tRNA without affecting tRNA carrying an amino acid. This sample labelled "Experimental" in Table 6 , incorporated 43 percent of the amount of tritiated leucine incorporated by the

Control RNA sample. This may indicate that 43 percent of the RNA was aminoacylated since, as mentioned previously, 91

Table 6 . Effects of various agents on the aminoacylation reaction. Experimental details are given in the text.

H-leucine Sample First 50 ul Second 50 ul cpm Percent Addition Addition accepted

Control h 2° h 2° 70,352 100

Experimental 0.0lMNaI0 4 0.035MNaHS03 30,222 43

Iodine NaI0 4 +NaHS0 3 Nal0 4 +NaHS0 3 106,231 151 (pre-mixed) (pre-mixed)

10 0.01M NalO. h 2° 5,800

1 0 . 0.01M NalO, h 2° 8,153 12

Blank 5,692 (no RNA) 92

Morris (1966) found that the charging reaction could displace amino acids already present on tRNAs with amino acids present in the charging mixture without prior stripping.

Figure 9 shows that this can also happen under my conditions of charging.

Another possible explanation for this 43 percent level of incorporation is that the reaction products of sodium periodate and sodium hydrogen sulfite somehow lower the efficiency of the arainoacylation reaction. To test this possibility, equal volumes of sodium periodate and sodium hydrogen sulfite were mixed and allowed to react for 10 minutes at 37 degrees. This reaction mixture acquires a brown color due to the production of iodine. Fifty micro­ liters of this mixture was added to fifty microliters of

RNA and allowed to react 15 minutes at 37 degrees. An additional 50 microliters of iodine solution was then added to the mixture to duplicate the procedure used in other experiments in this series, and incubation continued for an additional 10 minutes. These fifty microliter additions are added in two stages to compensate for any diluting effects these additions might have. The RNA treated in this way was then aminoacylated as described. This sample labelled "Iodine" in Table 6 incorporated

151 percent of the control counts. The basis for this 93

stimulation is unclear. It is certain, however, that the

reaction products of periodate and sodium hydrogen sulfite do

not inhibit the aminoacylation reaction.

The effects of iodate on the aminoacylation reaction

were tested by mixing sodium iodate with RNA and incubating.

Fifty microliters of water was added and incubation con­

tinued for 10 minutes. The mixture was then aminoacylated.

This sample, labelled "IO3 '' in Table 6 incorporated 8

percent of the control activity and 19 percent (8 percent s-

43 percent) of the Experimental activity. At the concen­

tration used, iodate inhibits the charging reaction.

A final experiment to determine if periodate itself

inhibits the charging reaction was performed using 0.01 M

sodium periodate. The method used was identical to that

described in the preceding paragraph, except that periodate

was substituted for iodate. The results of this experiment

are labelled "IOV1 in Table 6 . This experiment shows that periodate inhibits the charging reaction to a greater extent

than would be predicted on the basis of RNA oxidation alone.

Periodate reduced the amount of tritiated leucine accepted

to 12 percent of the control level and to 28 percent

(12 percent ^ 43 percent) of the Experimental activity.

The basis of the iodate and periodate inhibition of amino-

acylation is unclear, but these anions must be removed from

the RNA sample if complete aminoacylation is to be achieved. 94

The experimental scheme as described in the Materials and Methods separates and removes periodate and its reaction products from the RNA prior to aminoacylation. This method consists of periodate treatment for 15 minutes followed by reaction of the periodate with sodium hydrogen sulfite to produce iodine. The RNA is then precipitated with ethanol.

This procedure effectively separates iodine from the RNA since iodine is over 700 times more soluble in ethanol than in water. The treated samples are centrifuged and the supernatant discarded. The RNA was then stripped of amino acids by high pH incubation, precipitated and then amino- acylated.

Kinetics of Periodate Oxidation

To determine the length of periodate treatment needed to completely oxidize the tRNA, a fully stripped RNA prepa­ ration was divided into aliquots and treated with sodium periodate for varying periods of time. The control tube received only water. At intervals, sodium hydrogen sulfite was added and allowed to react. The RNA was then amino- acylated with tritiated leucine. The results are presented in Table 7. It is apparent that under the conditions used, periodate oxidation occurs very rapidly. Within two minutes the accepting activity dropped to less than 10 percent of the control value. This may reflect the great excess 95

Table 7. Kinetics of periodate oxidation

Minutes in periodate_____ cpm accepted_____ percent of control

Control (0 min.) 5,276 100

2 484 9

5 550 10

10 476 9

15 430 8

Blank (no RNA) 266 5 96 of periodate over the RNA in the mixture. No further

decrease in amino acid accepting activity was observed over

the fifteen minutes of periodate treatment.

In a similar experiment, Morris (1966) observed an

80 percent decrease in tritiated leucine accepting acti­

vity for 5 minutes (first sample taken) of periodate

oxidation. Further incubations showed a decrease in leucine

accepting capacity to 10 percent of the control level in

15 minutes. No further decrease in accepting capacity was

observed even after 25 minutes of treatment (final sample).

Even though my results show extremely rapid periodate

oxidation, all samples were subjected to periodate oxidation

for 15 minutes at 37°C in 5x10” M (final concentration)

sodium periodate.

Aminoacylation Levels

A primary objective of this work is to determine the

levels of aminoacylation of leucyl tRNA during balanced

growth and during leucine starvation. These parameters

have been determined for two strains of Bacillus subtilis

-- 3CW503 (a W23 strain) and BC29 (a 168 strain).

The data for aminoacylation levels of BCW503 during

balanced growth and during leucine starvation are summarized

in Table 8 . The results of individual experiments to

determine aminoacylation levels are presented in the Appendix

in Tables 11 through 14. 97

Table 8 . BCW503 Aminoacylation Levels

Experiment Mean cpm incorporated (std. error) Aininoacylation Number + 1 0 4 - 1 0 4 Level

1 +Leucine 3,685 (177) 6,445 (288) .57 -Leucine 744 (76) 4,734 (368) .16

2 tLeucine 2,467 (247) 2,038 (425) 1 . 2 1 -Leucine 1,039 (164) 3,789 (356) .27

3 +Leucine 5,021 (694) 5,078 (290) . 99 -Leucine 1,749 (216) 5,966 (466) .29

4 +Leucine 1,971 (1,178) 1,776 (991) 1 . 1 1 -Leucine 914 (594) 3,375 (348) . 27

+Leucine Mean: 0.97 (.14) -Leucine Mean: 0.25 (.03) 98

During balanced growth of BCW503, the mean level of aminoacylation for leucyl transfer RNA is 0.97 with a standard error of 0.14. The 95 percent confidence interval for the sample mean is defined (Li, 1964) by the sample mean, plus or minus the quantity 1.96 times the standard error. The value 1.96 is the 2.5 percent value of the

Student's t_-distribution used to test the equality of sample means at the 95 percent level of significance. The

95 percent confidence interval for aminoacylation in

BCW503 ranges from 0.69 to 1.25. Inspection of Table 8 reveals that the aminoacylation level in Experiment 1 of 0.57 falls outside the 95 percent confidence level.

When a new mean value for balanced growth aminoacylation is computed, omitting this low value, the mean level of aminoacylation becomes 1.10 with a standard error of 0.06.

The 95 percent confidence interval is now 0.98 to 1.23.

Thus, during balanced growth of BCW503, 98 percent or more of the leucyl tRNA is aminoacylated either with leucine or with some other substance that protects the tRNA from periodate oxidation.

During leucine starvation, the level of aminoacylation for leucyl tRNA in BCW503 falls to a level of 0.25 with a

95 percent confidence interval of 0.19 to 0.31. Therefore, leucine starvation of BCW503 results in a drop in 99 aminoacylation of leucyl tRNA of over 70 percent, from the balanced growth level in excess of 98 percent to a star­ vation level of 25 percent.

The datd for aminoacylation levels of BC29 tRNA during balanced growth and during leucine starvation are summarized in Table 9. The results of individual experiments to determine aminoacylation levels are presented in the

Appendix, Tables 15 through 18. Table 9 shows that the levels of aminoacylation for BC29 leucyl tRNA during bal­ anced growth are lower than comparable values for BCW503.

The mean level of leucyl tRNA aminoacylation for BC29 in balanced growth is 0.63 with a standard error of 0.03, indicating that the 95 percent confidence interval is

0.58 to 0.68.

During leucine starvation of BC29, the level of leucyl tRNA aminoacylation drops to 26 percent. This level of aminoacylation is comparable to the 25 percent level seen during leucine starvation of BCW503. Thus, while the levels of leucyl tRNA aminoacylation for BCW503 and BC29 differ by almost 35 percent during balanced growth; during leucine starvation, both strains contain leucyl tRNA aminoacylated to the same degree.

Sepharose 43 Chromatography of Leucyl tRNA

A second major objective of this work was to determine the charging profiles, or levels of aminoacylation, of various leucyl isoaccepting tRNAs during balanced growth and during 100

Table 9. BC2 9 Aminoacylation levels

Experiment Mean cpm incorporated (std. error) Aminoacylation Number +10^ Level 4 4

1 +Leucine 1,397 (166) 2 , 1 2 0 (442) 0 . 6 6 -Leucine 630 (116) 2,687 (1 1 2 ) 0.23

2 +Leucine 13,870 (307) 20,409 (651) 0 . 6 8 -Leucine 3,686 (2 2 2 ) 2 , 2 2 1 (876) 0 . 2 2

3 +Leucine 6,813 (586) 12,254 (134) 0. 56 -Leucine 3,095 (1,336) 14,080 (863) 0 . 2 2

4 tLeucine 6,915 (539) 11,083 (542) 0.62 -Leucine 4,755 (1,108) 13,161 (501) 0. 36

+Leucine Mean: 0.63 (.03) -Leucine Mean: 0.26 (.04) 101 leucine starvation. Sepharose 4B column chromatography using a reversed (2.0 M decreasing) ammonium sulfate gradient was used to separate peaks of tritiated leucine accepting activity (Holmes et_ al., 1976). In addition to determining tritiated leucine accepting activity, effluent fractions were analyzed for absorbance at 260 nanometers and for refractive index. The absorbance at

260 nanometers indexes the presence of nucleic acid. Refrac­ tive index is an intensive property and when specified with temperature can be used to determine the concentration of ammonium sulfate at which a given isoaccepting peak elutes from the column.

In the course of preliminary experiments with the columns a large peak, eluting near the void volume, was observed. This peak was assumed to be a peak of radio­ activity that was not specifically associated with tRNA.

To check on this hypothesis an "aminoacylation" reaction was carried out using a charging mixture complete in every respect, except that the aminoacyl tRNA ligase that was added to the reaction mixture had previously been boiled for thirty minutes to completely denature it. This "charging reaction" produced an increase in TCA precipitable acti­ vity of less than ten percent. The results of this experi­ ment are presented in Figure 10. Inspection of this figure reveals a single peak of tritium activity (4,5-% leucine) between fractions 21 and 29 inclusive. The refractive 102

Figure 10.

Sepharose 4B chromatography of leucine tRNA "arainoacylated" with inactive synthetase. Prior to reaction, the synthetase was boiled for thirty minutes to completely denature it. CPM(xlO“ 200 120 0 4 o- 20 Vv oo INACTIVE SYNTHETASE APE NUMBER SAMPLE 0 4 260 A 0 6 oi 80 100 IUE 10FIGURE 260 A 103 0.9 -0 -1.3 0.4 -0 .5 -0 - .7 -0 - - - .3 -0 0.6 0.8 0.2 0.1 1.3300 1.3500 1.3600 1.3700 1.3200 1.3400 104

index of these fractions ranges from 1.3695 (fraction 20)

to 1.3692 (fraction 30). The increase in refractive index

at fraction 25 (n^,: 1.3735) was thought to be due to the

high density of the suspending TPC plus 2 M ammonium

sulfate used to load the charged RNA onto the column. This

suspending buffer had glucose added to increase its density

and to facilitate the addition of the charged tRNA to the

column in a small volume. The integrity of this density peak gave credence to the possibility that small molecules

other than glucose -- perhaps even tritiated leucine --

might pass through the Sepharose 4B column in the void

volume without significantly diffusing out of the glucose

density band.

To test this possibility, fraction 18 from Figure 13

(the void volume peak) was examined for absorbance at 260

nanometers. The sample was then dialyzed against a total

of three one liter changes of distilled water with eight

hours for each change. The initial absorbance at 260 nano­

meters was 1.208 and the tritium activity was 3775 disin­

tegrations per minute. If the radioactivity was not

covalently associated with a macromolecule, then the radio­

activity should decrease after dialysis. After dialysis

the tritium activity had decreased to a background level of

83 disintegrations per minute, and the absorbance at 260

nanometers had decreased to 0.079. The conclusion is that 105 the void volume peak of radioactivity and 260 nanometer absorbance represents tritiated leucine and glucose, plus other small molecules that absorb at 260 nanometers. The ultraviolet absorbance can not be entirely attributed to glucose, since a solution of TPC and 2 molar ammonium sulfate, saturated with glucose, showed an absorbance of

0.133. The glucose, tritiated leucine and small molecules migrate through the column without diffusing, while the transfer RNA remains bound to the Sepharose 4B. The RNA begins to elute at fraction 47 as indicated by the increase in absorbance at 260 nanometers. The RNA continues to elute to fraction 84.

Since the only peak of radioactivity occurred in a region well separated from the RNA peak; since the glucose density band remained intact in its passage through the

Sepharose 4B column; and since the synthetase was inactivated by boiling; the first peak (Vv) is concluded to represent non-specific association of tritiated leucine with some component present in the loading buffer and is not con­ sidered to be leucine isoaccepting RNA.

Figure 11 is a plot of refractive index, absorbance at 260 nanometers and tritiated leucine accepting acti­ vity for a sample of BCW503 RNA isolated from cells in balanced growth. Prior to aminoacylation, the transfer

RNA was periodate oxidized and then stripped of amino acids 106

F i g u r e 11.

Sepharose 4B chromatography of leucine tRNA isolated from cells of BCW503 in balanced growth. Prior to amino­ acylation, the tRNA was periodate oxidized and then stripped of amino acids by pH 8 incubation. CPM(xlO~ 0 3 20 20 40 C53 BALANCED GROWTHBCW503 SAMPLE NUMBER 60 260 0 8 100 0 6 2 A ■0.5 1.3200 ■ 0.6 -0.7 • ■0.9 • -0.3 ■0.4 0.8 0.1 0.2 107 IUE 11 FIGURE •1.3300 ■1.3400 ■ 1.3500 -1.3600 1.3700 108 by incubation at pH 8 . Three peaks of leucine accepting activity are associated with the &-2b0 Pea^s* The peaks of tritiated leucine accepting activity were labelled as follows: peak A occurs in fractions 41 to 50 inclusive

(up: 1.3572-1.3513); peak B occurs in fractions 53 through

57 (nD : 1.3500-1.3481); peak C occurs in fractions 59 through 63 (nD : 1.3461-1.3458). The relative areas for the three tRNA peaks were determined by weighing paper cut­ outs. They are, in order: peak A, 55 percent of the total

tritiated leucine accepting activity; peak B, 17 percent; and peak C, 28 percent.

Figure 12 presents the results of a Sepharose 4B

column loaded with RNA isolated from leucine starved BCW503.

As in balanced growth, three peaks of leucine accepting activity were resolved. Peak A (np: 1.3556-1.3524) con­ tained 58 percent of the total leucine accepting activity; peak B (np: 1.3509-1.3489) contained 20 percent of the

total leucine accepting activity; and peak C (nD : 1.3482-

1.3468) contained 22 percent of the total tritiated leucine

accepting activity.

The proportions of the total leucine bound by each

of the three leucyl tRNA peaks remain constant in the presence or in the absence of leucine in the growth medium

even though the total aminoacylation levels are lower when

leucine is absent. 109

Figure 12.

Sepharose 4E chromatography of leucine tRNA isolated from cells of BCW503 after leucine starvation for two generation time equivalents. Prior to aminoacylation, the tRNA was periodate oxidized and then stripped of nascent amino acids by high pH incubation. CPM(xlO') 240 160 0 8 20 C53 ECN STARVATION LEUCINE BCW503 APE NUMBER SAMPLE 0 4 oo 60 0 6 2 A 80 IUE 12 FIGURE 260 A 110 1.6 - r -1.5 -1.4 -1.3 - - - 0.9 -0 - .7 0 - - .5 0 - .3 0 - .4 0 - - 0.8 1.0 1.2 1 . 1 0.6 .1 0 0.2

nl ^ 1.3200 ^ -1.3300 0 0 4 .3 -1 -1.3500 -1.3600 1.3800 1.3700 Ill

Figures 13 and 14 present the results of Sepharose 4B chromatography on two different samples of RNA isolated from growing cells of BC29. The RNA sample in Figure 13 was obtained from cells of BC29 in balanced growth in minimal medium at a cell density comparable to that in Figures 11,

12 and 15. The RNA sample in Figure 14 is from cells of

BC29 in exponential growth in Trypticase Soy Broth (TSB).

The growth conditions in Figure 14 are similar to those used by Tockman and Void (1977) to obtain cells in logarithmic growth. Their procedure was to suspend 10^® heat shocked spores in one liter of tryptone-yeast broth and to allow growth for three hours. The procedure used to obtain the cells for which the RNA profile is shown in Figure 14 was to dilute an overnight culture of BC29 1:10 in one liter of pre-warmed TSB and to allow growth to proceed for four hours before harvesting the RNA. Consequently, the cell density is higher and the medium more complex for the RNA sample obtained and shown in Figure 14 than for the RNA samples shown in Figures 11 through 13 and 15.

There are differences between Figures 13 and 14. The most obvious is the presence of a fourth peak of leucine accepting activity in exponentially growing cells (Figure 14) that is not present in cells in balanced growth (Figure 13).

The three peaks in Figure 13 are as follows: peak A

(nD : 1.3560-1.3524) contained 62 percent of the total 112

Figure 13.

Sepharose 4B chromatography of leucine tRNA isolated from cells of BC29 in balanced growth. Prior to amino­ acylation the tRNA was periodate oxidized and then stripped of amino acids by high pH incubation. CPM(xlO') 0 4 2 160 80 o — — VvN 20 C9 AACD GROWTH BALANCED BC29 0 4 APE UBR IUE 13 FIGURE NUMBER SAMPLE 080 60 A260 1.2 T 0.9 -0 0. *-|.3200 .6 -0 .7 -0 - 0.3 -0 .4 -0 .5 -0 - 0.8 0.2

100 r-1.3700 -1.3600 -1.3500 11 1.3400 .3300 3 114

Figure 14.

Sepharose 4B chromatography of leucine tRNA isolated from cells of BC29 in exponential growth (see text). Prior to aminoacylation, the tRNA was periodate oxidized and then stripped of amino acids by pH 8 incubation. J 0 CPM (xlO“ 30 20 040 20 C9 XOETA GROWTH EXPONENTIALBC29 A260 ' b « o' q o SAMPLENUMBER 9 60 i

oo 80 oooc xooooooR x) c x) xooooooR oooc 100 A260 IUE 14 FIGURE r-0.5 -0.4 -0.3 - 115 0.2 0.1 - r D L 1.3200 -1.3600 -1.3500 -1.3300 -1.3400 1.3700 116 tritiated leucine accepting activity; peak B (nD : 1.3507-

1.3481) contained 16 percent; and peak C (nD : 1.3481-

1.3456) contained 22 percent of the total tritiated leucine activity.

Peaks A, 3, and C in Figure 14 correspond to peaks A,

B, and C in Figure 13 with respect to observed refractive index. These peaks are: peak A (np: 1.3549-1.3517) con­ tained 69 percent of the total tritiated leucine accepting activity; peak B (np: 1.3496-1.3476) contained 6 percent of the total; and peak C (np: 1.3468-1.3452) contained

19 percent of the total tritiated leucine activity. The unique peak D, found only in this exponentially growing

RNA sample occurred at refractive indeces 1.3442-1.3440 and contained 6 percent of the total tritiated leucine accepting activity.

Figure 15 presents the results of a Sepharose 43 column loaded with RNA isolated from leucine starved cells of BC29. As in balanced growth, during leucine starvation three peaks of leucine accepting activity are apparent.

Peak A (np: 1.3554-1.3526) contained 56 percent of the total leucine accepting activity; peak B (np: 1.3506-

1.3488) contained 21 percent of the total; and peak C

(nD : 1.3481-1.3465) contained 23 percent of the total tritiated leucine accepting activity. 117

Figure 15.

Sepharose 4B chromatography of leucine tRNA isolated from cells of BC29 after leucine starvation for two genera­

tion time equivalents. Prior to aminoacylation, the tRNA

was periodate oxidized and then stripped of nascent amino

acids by high pH incubation. CPM(xlO” 30 20 40 C9 LEUCINE STARVATIONBC29 \ o o CD SAMPLENUMBER A260 60 8020 100 A 260 0.l L 0.9 -0 -0.5 *-1.3200 -0.6 -1.3300 -0.8 -0.7 -0.4 - -0.3 0.2 118 120 r 1.3700 r -1.3500 -1.3400 IUE 15 FIGURE .3600 119

In BC29, as in BCVJ503, during balanced growth or during

leucine starvation; each of the three leucyl tRNAs binds a constant proportion of the total RNA associated tritiated

leucine, even though the overall level of aminoacylation is

lower during leucine starvation.

Table 10 summarizes the data presented in Figures 11

through 15 in order to facilitate comparisons. There are

three conclusions to be drawn from an inspection of this

table. First, during balanced growth or during leucine

starvation, different peaks (A, B, and C) contribute

different proportions to the total level of aminoacylation.

Peak A shows the greatest leucine accepting activity and

hence contributes the largest amount to the overall level

of aminoacylation. Peaks B and C contribute lower and

approximately equal proportions to the total level of amino­

acylation.

Secondly, for any given peak, the contribution of that peak to the total level of aminoacylation is relatively

constant for both cell lines in the presence or absence of

leucine. Because of the nature of the methods used, some

deviation is expected even for duplicate RNA samples. A

second column run with RNA isolated from BC29 in balanced

growth showed that peak A bound 70 percent, peak B bound

11 percent and peak C bound 19 percent of the total RNA

associated leucine. Table 10. Sepharose 4B Chromatography Summary

% Total ^H-Activity Organism/Condition Peak A Peak B Peak C Peak D

BCW503+Leucine^ 55 17 28 —

BCW503-Leucine^ 58 20 22

BC29+Leucine^ 62 16 22

BC29 Logarithmic^ 69 6 19

2 BC29-Leucine 56 21 23

Mean (SX)4; 58 (2) 19 (1) 24 (1)

1. Cells in balanced growth in minimal medium containing leucine

2. Balanced growth cells starved for two generations in minimal medium

3. Cells beginning logarithmic growth in Trypticase Soy Broth 120 4. Excluding BC29 logarithmic growth samples 121

Finally, a fourth peak of leucine accepting activity is seen in BC29 only during exponential growth in rich medium* The appearance of this peak is associated with a decrease in peak B„ This fourth peak did not appear in either of two columns run with BC29 balanced growth RNA, nor did it appear in the single column run with BC29 leucine starved RNA. DISCUSSION

This work had two major objectives. The first was to determine the levels of aminoacylation of bulk leucyl tRNA isolated from cells of Bacillus subtilis during bal­ anced growth and to determine how these levels change during leucine starvation. The second objective was to separate,

in so far as possible, individual leucine accepting tRNAs using Sepharose 4B column chromatography and to determine

how changes in these individual leucine isoacceptors reflect

changes in the overall level of leucine tRNA aminoacylation.

Both of these objectives have been achieved.

Periodate Treatment

In order to determine what fraction of the tRNA is

aminoacylated in vivo, it is necessary to inactivate those tRNAs that are not aminoacylated. One of the established methods for the inactivation of non-aminoacylated (uncharged)

tRNAs is periodate oxidation (Hecht et al., 1959). Iodate

-- a reduction product of periodate -- can inhibit amino­

acylation of tRNA by aminoacyl tRNA synthetase (Ri2 zino and

Freundlich, 1975). I have also found that periodate at

similar concentrations inhibits the aminoacylation of

122 123

Bacillus subtilis leucyl tRNA to a greater extent than would be predicted on the basis of oxidation of uncharged tRNA.

Consequently, it is necessary to remove both of these inter­ fering anions from the reaction mixture prior to amino­ acylation.

Overnight dialysis has been employed by some authors

(Tockman and Void, 1976) to remove interfering iodate, but this method is time consuming and exposes the tRNA to pos­ sible attack by RNase. This would significantly affect findings on aminoacylation levels. I have taken advantage of the reaction of sodium hydrogen sulfite on iodate and periodate to remove these anions. This reaction is much faster than dialysis. It is complete in five minutes at

37°C. The product of this reaction is iodine, which is soluble at 0.03 grams per 100 ml water and 20.5 grams per

100 ml ethanol. Thus, the ethanol precipitation of the periodate oxidized tRNA effectively separates the RNA from the iodine and the iodine is then discarded in the super­ natant .

I feel that this method is superior to dialysis, since it is much less laborious and does not require large amounts of materials (such as dialysis buffer). This method has the added advantages over dialysis of minimizing the exposure of the RNA sample to RNase and allowing periodate treatment, high pH de-aminoacylation and aminoacylation -- all in the same tube. 124

Bulk Aminoacylation Levels

Two strains^of 3acillus subtilis have been compared.

The results of the bulk aminoacylation study show that in

BCW503 (a W23 strain) the mean balanced growth araino- acylation level is 97 percent. During leucine starvation, this aminoacylation level drops to 25 percent. In contrast, the 168 strain (BC29) shows a lower mean leucyl tRNA amino­ acylation level equal to 63 percent in balanced growth and

26 percent during leucine starvation.

There are two obvious conclusions: (1) during balanced growth, different strains of Bacillus subtilis aminoacylate their transfer RNAs to different degrees and (2) during leucine starvation, both strains show decreases -- to approxi­ mately 25 percent -- in bulk aminoacylation levels, thus some fraction of the leucyl tRNA always remains aminoacylated.

The mean level of 63 percent aminoacylation for the

168 strain (BC29) is in good agreement with the value of

Tockman and Void (1977). They reported 58 percent amino­ acylation of a trpC2 mutant of strain 168. During stationary phase, this level of aminoacylation increased to 62 percent.

While their cells may have been growing logarithmically, their methods, as described in the Introduction, make it unlikely that they were in balanced growth -- a physiological state characterized by constant rates of DNA, RNA, and protein synthesis. Still, their results showed that within 125 three hours of outgrowth from spores, RNA achieved a level of aminoacylation comparable to that in balanced growth. In addition, this aminoacylation level was maintained at 62 percent during stationary phase.

The differences in aminoacylation levels during balanced growth between the two strains I have used suggests that there are strain differences in aminoacylation levels. This obser­ vation has some precedent in work with E. coli. Yegian et al. (1966) found a leucyl tRNA aminoacylation level of 97 percent for cells of a stringent strain of E_. coli C600

(Thr-, Leu-, Thia-) in exponential growth, whereas the level in a relaxed strain, K-12, W1305 (Met-, Leu-) was 88 percent in exponential growth. During leucine starvation, these aminoacylation levels decreased to 20 percent in the strin­ gent strain (C600) and to 13 percent in the relaxed strain

(K-12, VJ1305). Morris and De Moss (1966) found a value of

70 percent aminoacylation for leucyl tRNA of cells of the stringent K-12 strain, 2961 (Leu-, Pro-, Thr-, Thia-) during exponential growth and 90 percent for cells of the wild type. During leucine starvation, this level dropped to 40 percent.

The preceding variations of values could indicate differences in aminoacylation levels among different stringent cell lines of E. coli K-12. However, it is difficult to compare directly the values obtained by different groups using different methods. Factors that can contribute to 126 the observed differences include the fact that some de- aminoacylation of tRNA does occur during isolation (Table 3) and different isolation schemes may differentially affect tRNAs isolated under different growth conditions. Further­ more, Morris (1966) did not strip the tRNA prior to amino­ acylation, while Yegian ert al. (1966) did.

Both periodate and and iodate (produced during periodate treatment of uncharged tRNA) can inhibit the aminoacylation reaction (Rizzino and Freundlich, 1975). Yegian et_ al. did not remove the periodate and the solubility of periodate and iodate in ethanol and ethanol-acetate makes it likely that significant amounts remained in the mixture prior to charging. Morris attempted to remove sodium periodate by precipitating it as the potassium salt. (I have attempted this method, but have had no success with it.) No attempt was made to remove residual iodate. Void's method (Tockman and Void, 1977) used overnight dialysis to remove residual iodate after the periodate had been reduced by oxidation of excess ethylene glycol. No mention is made that any RNase inhibitor was present in the dialysis buffer and some RNA breakdown by RNase is likely to have occurred.

Void (Tockman and Void, 1977) has stated that "inter­ fering substances" such as polysaccharides could interfere with effective periodate oxidation. Neither of the groups working with B. coli leucyl tRNA state that any attempt 127

was made to remove these interfering substances. In the

work that I have done (Table 7) periodate oxidation is quite

effective in oxidizing tRNA without removal of interfering

substances.

Table 4 shows that the relative amount of tRNA can vary

from batch to batch making determinations of accepting activty per milligram of RNA difficult to interpret unless the precent

4^ RNA is determined. Differences in cell density could

also influence the level of tRNA aminoacylation within a

given strain.

The growth medium employed can influence aminoacylation

levels. Morris (1966) found that in 0.4 percent glycerol

(135 minute generation time) the leucine aminoacylation level

of the wild type of E. coli was 76 percent. In 0.4 percent

glucose (65 minute generation time) or in nutrient broth-

yeast extract (32 minute generation time) this level

increases to 90 and 93 percent, respectively.

All of the factors mentioned above make work between

different groups difficult to compare. However, my work

using identical methods and conditions makes the conclusion

more certain that there are real differences in aminoacylation

levels between 3C29 and BCW503 during balanced growth.

These differences in aminoacylation levels may be strain

specific, that is all 168 strains may aminoacylate their

leucyl tRNA to 63 percent during balanced growth. Alterna­

tively, these aminoacylation levels may reflect differences 128 within a given cell line. That is, some 168 mutants may aminoacylate their leucyl tRNA to 63 percent in balanced growth, while other 168 mutants might have significantly different levels of leucine tRNA aminoacylation. To dis­ tinguish these two possibilities, it would be necessary to determine leucine tRNA aminoacylation levels for a variety of 168 mutants. If all 168 mutants examined aminoacylated their leucine tRNA to 63 percent during balanced growth and all W23 strains examined aminoacylated their leucyl tRNA to 97 percent, it would be concluded that levels of leucine tRNA aminoacylation reflect another difference between these two strains. This finding could then be added to the list of known differences between 168 and W23 strains. This list includes differences in cell wall com­ position and susceptibility to phage (Glaser et_ al., 1966), control of chromosome replication (Yoshikawa and Sueoka,

1963) and efficiency of genetic exchange (Dubnau et al., 1969).

While the levels of leucine tRNA aminoacylation are different in balanced growth of BC29 and BCW503, during leucine starvation the leucine tRNA aminoacylation levels for the two strains are identical. In BCV7503, the level of aminoacylation drops to 25 percent while the starvation level in BC29 is 26 percent. An unpaired t-test revealed that these means are not significantly different at the 129

5 percent level of significance. Both of these strains are stringent for RNA control (Thomas and Copeland, 1973).

My values for aminoacylation levels for two strains of

Bacillus subtilis during leucine starvation are in good agreement with those of Yegian et al. (1966) who found a level of 20 percent aminoacylation of leucine tRNA during leucine starvation of a stringent strain of E. coli and

13 percent for a relaxed strain. Morris' (Morris and DeMoss,

1966) value of 40 percent aminoacylation during leucine starvation is higher than mine or that of Yegian et_ al.

This higher value may reflect a difference in the methods used to achieve leucine starvation. Morris' method was to add a limiting amount of leucine and to allow growth to continue until optical density had ceased to increase for one generation time. As stated in the Literature

Review, this slowing of growth may allow any of a number of other cellular regulatory systems to be perturbed in ways that might indirectly affect leucine tRNA aminoacyl- atinn. This higher level of aminoacylation may also reflect the tightness of the Leu- phenotype in K-12,

2961; since optical density never did actually plateau under Morris' conditions. Other factors that could con­ tribute to these observed differences in aminoacylation levels have been discussed above. 130

Two obvious questions arise with regard to my leucine

starvation aminoacylation levels: (1) Why don't the leucyl tRNA aminoacylation levels fall to aero during leucine starvation? and (2) Why do two organisms with different aminoacylation levels during balanced growth reduce these levels in such a way during leucine starvation so that both BCW503 and BC29 have the same level of leucyl tRNA arainoacylat ion?

Morris (1966) concluded that the 40 percent level of charged leucine tRNA that he found could be entirely attributed to a protein turnover level of 5 percent per hour.. Under his conditions, this was shown to be true.

Thomas and Copeland (197 3) have shown that during leucine

starvation of BC29 and BCW503 less than 10 percent of the proteins are labelled during the starvation. This indi­ cates that protein turnover does occur during leucine

starvation, and at a level comparable to that seen by

Morris.

Aminoacylation levels are determined on the basis of periodate resistance. It is assumed that tRNAs resistant to periodate oxidation are protected from oxidation by

their cognate amino acid. Yegian and co-workers (Yegian

et al., 1966; Yegian and Stent, 1969) found that during leucine

starvation between 10 and 20 percent of the total leucine 131 accepting tRNA contained an alkali stable protector that prevented periodate oxidation of that fraction of the leucine tRNA. As stated in the Literature Review, the identity of this protector remains unknown.

It is possible that during leucine starvation, Bacillus subtilis also protects some portion of the leucine tRNA with a stable protector. This possibility could be tested in the manner of Yegian and Stent (1969) as outlined on page 39 of the Literature Review. A sample of tRNA from leucine starved cells could be stripped by pH 8 incubation.

This should remove the amino acids from the tRNA, but should not affect the stable protector. The sample could then be oxidized with sodium periodate to inactivate all non- aminoacylated tRNAs. Then the sample could be charged with unlabelled leucine. If the protector behaves like that found by Yegian and Stent, this should result in replacement of the protector with unlabelled leucine. This sample would then be stripped, periodate oxidized, and then amino­ acylated with tritiated leucine. If the stable protector can be removed by the first (unlabelled leucine) amino­ acylation and replaced with unlabelled leucine, as was the case for the protector found by Yegian and Stent, then the second alkaline hydrolysis and subsequent periodate treatment should make the leucine tRNA incapable of accepting tritiated 132 leucine in the second aminoacylation reaction. If, on the other hand, this stable protection is due to an intrinsic structural feature of the tRNA (such as a 3'-deoxyadenosine moiety) the leucine tRNA should be unaffected and should accept the tritiated leucine in the second charging reaction.

An experiment of this type would reveal if a protector was responsible for some or all of the leucyl tRNA that remained aminoacylated (periodate resistant) after leucine starvation. It would also determine whether the protective action was due to a structural alteration of the tRNA or an addition to the 3 1-end of the tRNA molecule.

The second question raised by the finding of equal levels of leucine tRNA aminoacylation during leucine star­ vation is, "Why do two organisms with different amino­ acylation levels in balanced growth, exhibit the same level of leucine tRNA aminoacylation during leucine starvation?"

One possible explanation is that this finding is fortuitous and that investigations of leucine aminoacylation levels in other strains of B. subtilis would show that during leucine starvation different levels of leucine tRNA amino­ acylation are achieved. This explanation could only be resolved by further experimentation. However, the fact that Yegian and co-workers found a similar level of leucine tRNA aminoacylation during leucine starvation in E. coli suggests that the residual 25 percent level of leucine tRNA aminoacylation may be more than fortuitous. 133

Another possible explanation, for example, is that

during leucine starvation the turnover levels in BC29 and

BCW503 are equal and therefore their tRNAs are aminoacylated

to equal degrees, Thomas and Copeland (1973) did find

similar levels of protein turnover in the two strains. These

levels averaged under 10 percent. One might expect that

if 25 percent of the leucine tRNA is aminoacylated and if no single isoacceptor can be shown to be missing, then protein synthesis would be expected to continue at approxi­

mately 25 percent of the balanced growth level in BCW503

and 41 percent (26 percent divided by 63 percent) of the balanced growth level in BC29. This is not observed.

An intriguing explanation for the observed equality of

starvation aminoacylation levels is that the 25 percent

level of leucine tRNA aminoacylation represents a "sub­

threshold" level for protein synthesis. For example,

tRNA may need to be charged at 30 percent before protein

synthesis can begin. Copeland and Thomas (1973) found that

when leucine was added to leucine starved cells of BC29 or

BCW503, protein synthesis resumed with no detectable delay.

If a threshold level for protein synthesis exists it should be possible to test for it by growing cells of BC29 or

BCW503 in a leucine-limited chemostat. The addition of

leucine could be slowed and the level of aminoacylation

determined at various rates of leucine addition. When the 134

level of aminoacylation went below the threshold value necessary to maintain protein synthesis, the cells would

stop growth and be washed out of the chemostat because the

amount of leucine being added would be insufficient to

allow protein synthesis to take place.

Sepharose 43 Chromatography

The second major objective of this study was to separate,

in so far as possible, individual leucine accepting tRNAs

using Sepharose 4B column chromatography in order to deter­

mine how the aminoacylation levels of these individual

acceptors reflect the overall level of leucine tRNA amino­

acylation. I have been able to resolve three peaks of

leucine accepting activity for BC29 and BCW503 in balanced

growth. Three peaks are also detected during leucine star­

vation of both strains. During logarithmic growth of BC29,

a fourth peak of elucine accepting activity was detected,

in addition to the three peaks observed during balanced

growth. These peaks have been labelled, in order of their

elution from Sepharose 4B as peaks A, B, C, and D.

The method of comparison chosen emphasizes similarities

in peak contributions. This similarity becomes more apparent

if the contributions of each peak to the total leucine

accepting activity in both strains during balanced growth

and leucine starvation are averaged together (excluding the

results for exponentially growing 3C29). The mean 135 contribution for peak A is 58 percent with 95 percent confidence limits from 54 to 62 percent. Similarly, the mean contribution of peak B is 19 percent with confidence limits of 17 to 21 percent and the mean contribution for peak C is 24 percent with limits from 22 to 26 percent. An unpaired t_-test comparing peaks B and C showed that the mean contributions for these peaks are different at the 5 percent level of significance. The contribution by peak B is less than the contribution of peak C. I conclude that the con­ tribution of each peak to the total leucine tRNA aminoacylation level is unique and constant in both strains under both conditions -- balanced growth and leucine starvation.

Thus, although BCW503 and BC29 exhibit differences in the overall level of leucine tRNA aminoacylation, a given peak contributes a definite, constant percentage to the overall level. Furthermore, during leucine starvation in both strains, all leucine tRNA peaks decrease proportion­ ately with the overall decrease in leucine accepting activity.

No tRNA peak is spared or sacrificed to a greater extent than any other during leucine starvation.

As stated in the Literature Review, leucine, serine, and arginine share the distinction of being specified by the greatest number of codons. There are six codons for each of these amino acids and consequently six tRNAs for each. The presence of such-a large number of isoaccepting 136 tRNAs suggests that one or more could function as a regu­ latory signal without significantly affecting protein synthesis. RPC-5 chromatography can resolve six leucyl tRNAs from spores and from logarithmic cells of Bacillus subtilis trpC2 (Void and Minatogawa, 1972). Using Sepharose

43 chromatography, Holmes et_ al_. (1975) resolved four peaks of leucine accepting tRNA from commercial batches of

E. coli K-12 tRNA. Further characterization of these peaks by RPC-5 chromatography showed that peak A (the first peak to elute) contained tRNA-leu-2 and tRNA-leu-5. Peak

3 contained tRNA-leu-3, peak C contained tRNA-leu-1 and peak D contained tRNA-leu-5. It is not possible to compare my Sepharose 43 peaks directly with those of Holmes and co-workers since 3. subtilis and E. coli tRNAs elute from Sepharose 4B at different concentrations of ammonium sulfate. The isoacceptor compositions of ray peaks A, B,

C, and D would have to be determined on RPC-5 columns before these comparisons could be made. It is likely that one or more of my Sepharose 43 peaks consists of a mixture of two or more species of tRNA-leu. Consequently, if compensating changes in aminoacylation levels were to occur among t\vo isoacceptors within a given Sepharose 4B peak, these changes could not be detected. While no significant differences are seen in the tRNA profiles of balanced growing or leucine starved cells, the peak profile for BC29 in exponential growth consists of some extremes. Peaks 3 and C are lower than for any other cell sample. In addition, peak D is seen only during exponen­ tial growth of BC29. These differences suggest that the

BC29 exponential growth tRNA differs significantly from balanced growth and leucine starved tRNA. One wonders,

I'What is this difference due to?" One answer is that exponentially growing cells are only beginning to come out of stationary phase. The tRNA profiles, then reflect an

intermediate state between stationary phase and balanced growth with some of the characteristics of both. An extension of this idea is that during stationary phase in

BC29, peaks B and C decrease and peak D appears. There is

some precedent for leucine tRNA peak changes in different growth conditions in Bacillus subtilis. Void and Mina- togawa (1972) found tRNA-leu extracted from spores showed a higher amount of tRNA-leu-4 and tRNA-leu-6 and a lower amount of tRNA-leu-3 relative to tRNA extracted from log­ arithmically growing cells. This observation could be tested by growing cells of BC29 and BCW503 in minimal medium to stationary phase and examining the Sepharose 4B

leucine tRNA aminoacylation profile. 138

The present study was undertaken to determine the effects of leucine starvation on leucyl tRNA of Bacillus subtilis. Several effects unique to leucine tRNA were described in the Literature Review. For example, methyl deficient tRNA-leu exhibits an altered codon preference from equally efficient recognition of either poly UG or poly UC to more efficient recognition of poly UC (Capra and Peterkofsky, 1966), while methyl deficient tRNA-phe responds like fully methylated tRNA-phe to its cognate codons. Minor species of leucyl tRNA are needed for trans­ lation of exogenous messenger RNAs in normal mouse L-cells

(Zilberstein et al., 1976). Synonymous major species of tRNA-leu can not replace the minor species in translation.

T4 infection of E. coli results in destruction of one of the major host leucyl tRNAs (Sueoka and Kano-Sueoka, 1970) and replacement of the host tRNA-leu with a phage tRNA with a different codon specificity. The phage tRNAs are more effective in polypeptide synthesis using phage message than they are in translating host message (Scherberg and

Weiss, 1972). Furthermore, tRNAs are regulatory signals for the control of biosynthetic pathways. tRNAs participate in non-ribosomal reactions including cell wall synthesis

(Soffer, 1974) and priming of DNA synthesis (Harada et_ al. ,

1975). The list of reactions in which tRNA participates grows longer every year (Clark, 1977). 139

Leucine has a unique effect on chromosome replication in that during leucine starvation chromosomes of Bacillus subtilis do not replicate to completion (Copeland, 1969;

1971a, b; Copeland et al., 1976). This effect is unique to leucine starvation, since starvation for tryptophan, histidine, cysteine, arginine, and isoleucine results in completion of chromosomes (Phillips, 1974; Copeland et al.,

1976). Furthermore, a unique protector for leucine tRNA is produced during leucine starvation of E. coli (Yegian and Stent, 1969; Yegian et al., 1966). The unique effects of leucine on chromosome configuration, the large number of codons for leucine, coupled with the unique effects of various physiological conditions on tRNA-leu, the fact that tRNAs can a6t as primers for DNA synthesis, and the special protector for leucine tRNA produced during leucine star­ vation led to this investigation to determine how leucine starvation affected leucyl tRNAs in Bacillus subtilis. I have found differences in the balanced growth aminoacylation levels of leucine tRNA in two strains that differ in their control of chromosome replication in stationary phase

(Yoshikawa and Sueoka, 1961). During leucine starvation, both strains showed equivalent non-zero leucine tRNA amino­ acylation levels. Furthermore, all resolveable tRNAs showed identical profiles under both conditions in both strains. 140

This study shows that the mechanism of uncharging of leucine tRNA during leucine stairvation is random and not specific0 No leucine isoacceptor is sacrificed or spared over any other. These findings show that the peculiar effects of leucine starvation on the control of chromosome replication can not be attributed to a difference in tRNA-leu charging profiles resolveable by Sepharose 4B chromatography.

Some significant questions are raised by this study.

Is a leucine tRNA protector formed during leucine starvation in Bacillus subtilis? Does tRNA-leu or aminoacyl leucine- tRNA-leu act as a primer for DNA synthesis in Bacillus subtilis? Could the presence of a protector molecule on tRNA-leu abolish (temporarily or permanently) the capacity of tRNA-leu to prime DNA synthesis, or perhaps the synthesis of Okazaki fragments so that during leucine starvation, the synthesis of DNA is curtailed by a lack of functional primer molecules? Perhaps all leucine tRNA species parti­ cipate in this priming function. This hypothesis is not inconsistent with the results of this study.

Further work in this area could be undertaken to determine tRNA-leu profiles and aminoacylation levels in

Bacillus subtilis W23 and 168 in stationary phase. This condition is known to differentially affect the chromosome configuration in these two strains. Furthermore, it would be interesting and informative to know that if a tRNA-leu protector molecule is formed in Bacillus subtilis t what its identity is, and how it can be removed. These experiments could be expected to shed additional light on the effects of leucine starvation on B„ subtilis tRNAs and ultimately on the effects of leucine starvation on chromosome replication. SUMMARY

The levels of aminoacylation of leucine tRNAs have been

determined for two auxotrophic strains of Bacillus subtilis,

BC29 (a 168 strain) and BCW503 (a W23 strain). Sodium periodate was used to oxidize uncharged tRNAs in order to determine aminoacylation levels. Differences in overall

aminoacylation levels were observed. During balanced

growth in minimal medium, the level of leucine tRNA amino­

acylation is 97 percent in BCW503 and 63 percent in BC29.

During leucine starvation these levels drop to 25 percent

and 26 percent, respectively.

Sepharose 4B chromatography resolved three peaks of

leucine accepting activity. The contributions of each peak

to the total level of aminoacylation were unique and constant

for both strains under both conditions. The mean contri­ butions were: peak A (first to elute), 58 percent; peak B,

19 percent; and peak C, 24 percent of the total tRNAs resolved.

Four peaks were detected for exponentially growing

cells of 3C29 in Trypticase Soy broth. The peak contribu­

tions were: peak A, 69 percent; peak B, 6 percent; peak C,

19 percent; and peak D, 6 percent.

142 This work is unique in identifying differences in balanced growth aminoacylation levels in strains of Bacillus subtilis and in showing that, despite these differences, during leucine starvation aminoacylation of leucyl tRNAs decreases to the same, final, non-zero level in both strains.

Sepharose 43 chromatography showed identical profiles for both strains under both conditions, indicating that during balanced growth and during leucine starvation, each resolveable tRNA contributes a unique and constant amount to the the total level of leucine tRNA aminoacylation. APPENDIX A

Results of individual aminoacylation experiments for

BCW503 in balanced growth and during leucine starvation.

Experimental details are given in the text. These data are summarized in Table 8.

144 145

Table 11. Aminoacylation levels in BCW503. Experiment 1 leucine counts per minute (cpm) incorporated

Blank: 262

Balanced Growth Aminoacylation Periodate Treated Untreated Level

6,784 3,950 7,034 4,252 5,874 3,640 7,138 M e a n : 3,947 (177) 6,707 (288) -Blank 3,685 6,445 57%

Leucine Starved

Periodate Treated Untreated

1,040 5,610 1,156 5,162 1,036 5,282 794 3,830 M e a n : 1,006 (76) 4,996 (368) -Blank 744 4,734 16% 146

Table 12. Aminoacylation levels in BCW503. Experiment 2 ^H-leucine counts per minute (cpm) incorporated

Blank: 1,440

Balanced Growth Aminoacylation Periodate Treated Untreated Level

4,396 4,266 4,191 3,540 2,879 3,426 3,257 Mean: 3,907 (247) 3,478 (425) -Blank 2,467 2,038 121%

Leucine Starved

Periodate Treated Untreated

2,536 4,469 2,154 5,979 2,914 5,674 2,316 4,795 Mean: 2,480 (164) 5,230 (357) -Blank 1,040 3,790 27^ 147

Table 13. Aminoacylation levels in BCW503. Experiment 3 •^H-leucine counts per minute (cpm) incorporated

Blank: 619

Balanced Growth Aminoacylation Periodate Treated Untreated Level

7,530 5,699 6,011 5,062 5,118 4,269 5,961 Mean: 5,640 (694) 5,697 (290) -Blank 5,021 5,078 99%

Leucine Starved

Periodate Treated Untreated

2,038 5,479 2,628 6,872 2,840 6,297 1, 964 7,690 M e a n : 2,368 (216) 6,585 (466) -Blank 1,749 5,966 29% Table^W. Aminoacylation levels in BCW503. Experiment 4 H-leucine counts per minute (cpm) incorporated

Blank: 5,054

Balanced Growth Aminoacylat' Periodate Treated Untreated Level

6,533 9,264 8,807 5,275 5,947 6, 946 5,734 Mean: 7,024 (1,178) 6,830 (991) -Blank 1, 970 1,776 111%

Leucine Starved

Periodate Treated Untreated

4,945 8,340 5,292 9,070 7,621 7,874 6,015 5,496 Mean: 5,968 (514) 8,428 (348) -Blank 914 3,374 27% APPENDIX B

Results of individual aminoacylation experiments for

BC29 in balanced growth and during leucine starvation.

Experimental details are given in the text. These data are summarized in Table 9.

149 150

Table 15. Aminoacylation levels in BC29. Experiment 1 ^H-leucine counts per minute (cpm) incorporated

Balanced Growth

Blank: 2,266 Aminoacylation Periodate Treated Untreated Level

3,174 3,844 3,746 5,102 3,862 5,171 3,878 3,424 Mean (Sx) : 3,665 (166) 4,386 (442) -Blank 1,399 2,120 66%

Leucine Starved

Blank: 414

Periodate Treated Untreated

1,022 2,825 982 3, 220 808 3,320 1,364 3,026 Mean (Sx) 1,044 (116) 3,101 (112) -Blank 630 2,687 28% 151

Table 16. Aminoacylation levels in BC29. Experiment 2 ■^H-leucine counts per minute (cpm) incorporated

Balanced Growth

Blank: 3,464 Aminoacylation Periodate Treated Untreated Level

17,695 24,316 17,515 25,380 16,422 22,298 17,706 23,499 Mean: 17,334 (307) 23,873 (651) -Blank 13,870 20,409 68%

Leucine Starved

Blank: 2,397

Periodate Treated Untreated

2,294 3,159 3,184 2,796 4,459 3, 282 6,132 Mean (Sx) 2,883 (222) 4,618 (876) -Blank 486 2,221 22% 152

Table 17. Aminoacylation levels in BC2 9. Experiment 3 ■^H-leucine counts per minute (cpm) incorporated

Balanced Growth

Blank: 5,172 Aminoacylation Periodate Treated Untreated Level

11,845 12,608 14,756 13,085 18,968 10,402 18,557 M e a n : 11,985 (586) 17,427 (1,341) -Blank 6,813 12,255 56%

Leucine Starved

Blank: 5,172

Periodate Treated Untreated

10,938 19,658 6, 992 17,597 6,874 20,502 Mean: 8,268 (1,336) 19,252 (863) -Blank 3,096 14,080 22% 153

Table 18. Aminoacylation levels in BC29. Experiment 4 -^H-leucine counts per minute (cpm) incorporated

Balanced Growth:

Blank: 2,471 Aminoacylation Periodate Treated Untreated Level

8,128 14,562 10,724 13,270 9,579 12,152 9,112 14,232 M e a n : 9, 386 (539) 13,554 (542) -Blank 6, 915 11,083 62%

Leucine Starved

Blank: 2,471

Periodate Treated Untreated

4,786 15,274 5, 910 17,068 9,143 15,427 9, 064 14,759 Mean: 7,226 (1,108) 15,632 (499) -Blank 4,755 13,161 36% BIBLIOGRAPHY

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