typhimurium learn whether trnahis plays a role in the repression

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1 JOURNAL OF BACTERIOLOGY, Feb. 1972, p Copyright 1972 American Society for Microbiology Vol. 19, No. 2 Printed in U.S.A. Role of Histidine Transfer Ribonucleic Acid in Regulation of Synthesis of Histidyl-Transfer Ribonucleic Acid Synthetase of Salmonella typhimurium E. McGINNIS AND L. S. WILLIAMS Department of Biological Sciences, Purdue University, Lafayette, Indiana 4797 Received for publication 7 September 1971 The role of histidine transfer ribonucleic acid (trna) in repression of synthesis of histidyl-trna synthetase was examined in two strains of Salmonella typhimurium, one of which was a histidine trna (hisr) mutant possessing 52% of the wild-type (hisr+) histidine trna and a derepressed level of the histidine biosynthetic enzymes during histidine-unrestricted growth. Histidine-restricted growth caused a derepression of the rate of formation of histidyl-trna synthetase in both strains. In the case of the wild-type strain, addition of histidine to the derepressed culture caused a repression of synthesis of histidyl-trna synthetase for at least one generation of growth. In contrast, when histidine was restored to the derepressed hisr mutant culture, synthesis of histidyl-trna synthetase was continued at the initial derepressed rate. These results suggest that histidine must be attached to histidine trna for repression of synthesis of histidyl-trna synthetase. Repression of synthesis has been reported for 6 of the 2 aminoacyl-transfer ribonucleic acid (trna) synthetases (6, 7, 12). Significantly, Williams and Neidhardt (12) reported that restricted growth of histidine auxotrophs and bradytrophs by histidine resulted in a derepression of the rate of formation of histidyltrna synthetase of Escherichia coli. Restoration of histidine to these derepressed cultures caused an immediate reduction in the rate of synthesis of this synthetase in a manner similar to that reported for repression of synthesis of the histidine biosynthetic enzymes. For the histidine biosynthetic enzymes, the results of recent studies involving histidine regulatory mutants of Salmonella typhimurium have indicated an involvement of histidine trna (trnahis) in the repression process (7-9). Silbert et al. (1) reported nonrepressibility of the synthesis of the histidine biosynthetic enzymes in trnahis mutants (hisr) even when grown in the presence of excess amounts of histidine. These results led to the concept that histidine must be acylated to trnahis for repression of the histidine operon. The present study was undertaken to examine regulation of synthesis of histidyl-trna synthetase in strains of S. typhimurium and to 55 learn whether trnahis plays a role in the repression of synthesis of this synthetase. In this paper, we report evidence that the rate of synthesis of histidyl-trna synthetase is regulated by a mechanism resembling repression. More importantly, we also report evidence that the synthesis of histidyl-trna synthetase is not repressible by histidine in a hisr mutant. The latter result suggests an involvement of trnahis in the regulation of synthesis of another histidine enzyme (i.e., histidyl-trna synthetase) as has been reported for the histidine biosynthetic enzymes. MATERIALS AND METHODS Organisms. Two strains of S. typhimurium, prototrophic for histidine, were kindly provided by Mark Levinthal. Histidine auxotrophs were derived from these strains by ethyl methane sulfonate mutagenesis as described by Gorini and Kaufman (4). The two strains used in the present study are SP1 (ara9 mete338 iivc41 stra149 his3179) and SP2 (ara9 mete338 hisr1223 stral49 his3178). Media and methods of cultivation. The minimal medium used in this study was the basal salts solution of Fraenkel and Neidhardt (3) supplemented with.4% glucose and.2% ammonium sulfate as carbon and nitrogen sources. All amino acids were the L isomer and were used at a concentration of 1

2 56 McGINNIS AND WILLIAMS J. BACTERIOL. ug/ml for unrestricted growth. For histidine restriction,.5 pg of glycyl-dl-histidine/ml was used. The rationale for the use of glycyl-dl-histidine, in contrast to limiting amounts of L-histidine, was to achieve a slow and continuous supply of histidine to the cells. Previous studies have shown that a severe histidine restriction was required to observe derepression of histidyl-trna synthetase formation (12). Thus, this dipeptide was used at a low concentration (.5 yg/ml) to cause a significant reduction of the histidine pool below that maintained by a prototrophic cell growing in minimal medium. Deuterium oxide medium was prepared by using deuterium oxide (1%) instead of water. Cells were grown aerobically in a rotary-action shaker-at 37 C. In all experiments, cells were grown overnight in unrestricted medium and transferred at the beginning of each experiment to fresh medium of similar composition after being washed several times with minimal medium. Before growing cells in deuterium oxide media for density shift experiments, they were pregrown for several days in this heavy medium. In making transfers from heavy to light medium and from histidine-unrestricted to histidine-restricted medium, the cultures were centrifuged and washed twice with minimal medium before suspension in fresh medium to initiate the experiments. Growth was measured by an increase in optical density at 42 nm with a 1-cm light path in a Zeiss PMQ II or Hitachi-Perkin-Elmer, model 111, spectrophotometer. Preparation of cell extracts. Cells were subjected to sonic treatment with a Branson sonifier as described by Chrispeels et al. (2). The protein content of extracts was determined by the method of Lowry et al. (5). Preparation of trna. Approximately 25 ml of cells were grown in unrestricted medium to an optical density (42 nm) of 1.5. The cells were rapidly chilled, and were centrifuged at to 4 C. The resulting pellet was resuspended in.5 M sodium acetate buffer (ph 5.5) containing.6 M KC1 and.1 M MgCl2. The cells were subjected to sonic treatment with a Branson sonifier as described by Williams and Freundlich (11). Phenol extraction, ethanol precipitation, and column fractionation were as described elsewhere (11). After column fractionation, the 1 M LiCl fractions were pooled, and the isolated trna was deacylated of amino acids by incubation for 1 hr in.1 M tris(hydroxymethyl)aminomethane-hydrochloride, ph 8.9, at 37 C. The RNA content was determined by absorbance at 26 nm, with 24 absorbancy units considered equal to 1 mg of RNA per ml. The trna samples were used in the standard attachment assay system with '4C-histidine as the amino acid substrate, as described elsewhere (2). Histidinol phosphate phosphatase assay. The method of Ames et al. (1) was used except for several modifications. The incubation mixture consisted of.4 ml of crude extract,.22 ml of.1 M triethanolamine-hcl (ph 7.5), and.2 ml of histidinol phosphate to give a final volume of.28 ml. The mixture was incubated at 37 C for 3 min. Ascorbic acid plus molybdate reagent was added to stop the reaction. The tubes were read at 82 nm against an enzyme and buffer blank without histidinol phosphate. Histidyl-tRNA synthetase assay. HistidyltRNA synthetase activity was determined by the method of Chrispeels et al. (2) with the exception that incubation was at 37 C for 5 min. All values are the average of three determinations. One unit of activity was defined as that which formed 1 pamole of product/hr. Specific activity was expressed as units per milligram of protein. Centrifugation in cesium chloride. All procedures were as described by Williams and Neidhardt (12). The equilibrium centrifugation was performed in a Spinco SW 5.1 rotor by use of a Spinco- Beckman model L4 ultracentrifuge. Measurement of the de novo rate of enzyme synthesis. The rate of synthesis of histidyl-trna synthetase was determined by methods already described (12). Source of chemicals. Uniformly labeled 14C-Lamino acids were obtained from New England Nuclear Corp. (Boston, Mass.) E. coli K-12 trna was purchased from General Biochemicals Corp. (Chagrin Falls, Ohio). Chloramphenicol, L-histidinol, and L-histidinol phosphate were purchased from Sigma Chemical Co. (St. Louis, Mo.); cesium chloride, from Pierce Chemical Co. (Rockford, Ill.); and glycyl-dl-histidine, from Mann Research Laboratory (New York, N.Y.). RESULTS Level of histidinol phosphate phosphatase of hisr+ and hisr mutant strains. The cells were grown exponentially in unrestricted medium, washed twice, and grown with limiting histidine (.5 pag of glycyl-dl-histidine per ml). Growth of the hisr+ strain and the hisr mutant in media supplemented with excess histidine (1 pg/ml), with.5 pg of glycyl-dl-histidine per ml, and with no histidine is shown in Fig. 1. As shown in Fig. 2, histidinol phosphate phosphatase formation was derepressed in both strains during histidine-restricted growth. Upon the addition of excess histidine (1,g/ml) to these derepressed cultures, there was an immediate repression of synthesis of this histidine biosynthetic enzyme in the hisr+ strain (Fig. 2). However, the hisr mutant exhibited the same derepressed level of this enzyme for one-half mass doubling (at 12 min) after the restoration of histidine to the culture (Fig. 2). After 1. mass doubling of the mutant culture (at 15 min), there was a 3 to 35% reduction of the specific activity of this enzyme; yet, after about 1.5 mass doublings in unrestricted medium (at 18 min), the level of this enzyme in the hisr mutant was six to seven times higher than the repressed level of the hisr+ strain (Fig. 2).

3 VOL. 19, 1972 CONTROL OF HISTIDYL-tRNA SYNTHETASE 57 E N z Cl) w -j C.) F TIME (MIN) FIG. 1. Effect of various concentrations of histidine on growth of Salmonella typhimurium strain SPJ and its derivative SP2, a hisr mutant. The cells were grown exponentially in unrestricted medium, washed twice, and transferred to three separate flasks. The growth rate was determined for both strains growing in media containing no histidine (, ),.5,g of glycyl-dl-histidine/ml (1, ), and 1,ug of histidine/ml (A, A). Closed symbols represent the hisr+ strain, and open symbols represent the hisr mutant. Histidine trna activity of the hisr+ and hisr mutant strains. The trna was isolated as described in Materials and Methods, and the activity was determined by the standard attachment assay system with 14C-histidine as the amino acid substrate. The results shown in Table 1 indicate that the hisr mutant possessed approximately 52% of the trnahis found for the wild-type strain. These results are similar to those reported by Silbert, Fink, and Ames (1) for another hisr mutant. Regulation of synthesis of histidyl-trna synthetase of hisr+ and hisr mutant strains. The findings (Table 1) that decreased trnahis in the mutant strain was accompanied by an apparent lack of repression of histidinol phosphate phosphatase (Fig. 2) led to an examination of the role of trnahis in the regulation of histidyl-trna synthetase formation. Both the hisr+ strain and the hisr mutant were grown in unrestricted medium. The cells were washed twice and transferred to histidine-limited medium; subsequently, each culture received 1 Ag of L-histidine per ml. When the cells were transferred to histidinerestricted medium, there was an immediate C derepression of the rate of synthesis of histidyl-trna synthetase in both strains (Fig. 3). However, the hisr mutant possessed a higher level of this enzyme even during unrestricted growth (Fig. 3). Upon the addition of histidine to the restricted culture, there was an immediate repression of histidyl-trna synthetase formation in the hisr+ strain for almost one generation of unrestricted growth (Fig. 3). Conversely, histidine had no repressive effect on the rate of synthesis of histidyl-trna synthetase of the hisr mutant strain (Fig. 3). In fact, even after a generation of unrestricted growth, the mutant strain maintained a relative differential rate of formation of histidyltrna synthetase which was several-fold greater than the rate observed for the wildtype strain (Fig. 3). These results (Fig. 3) indicate that histidyl-trna synthetase formation is regulated in S. typhimurium, and suggest that the synthesis of this synthetase is not TIME ( MIN) FIG. 2. Effect of histidine-restricted and -unrestricted growth on the level of histidinol phosphate phosphatase in the hisr mutant (SP2) and hisr+ (SP1) strains of Salmonella typhimurium. At zero time, cells were transferred from an unrestricted culture, after two washings in minimal medium, to a medium containing.5 jig of glycyl-dl-histidine/ml and 1 jig of all other required amino acids/ml. Samples were taken at 3-min intervals. Excess histidine (1 Ag/ml) was added at the time indicated by the arrow. The activity was determined for the hisr+ culture () and hisr mutant culture () and plotted as a function of time.

4 58 TABLE 1. Transfer RNA (trna) activity for histidine in a hisr+ strain, SPI, and a hisr mutant, SP2a Quantity ConsprAcceptance Source of of trna Counts per activity nmn per mg relative trna assayed of trna to wild (mug) type (%) SPi 1. 6, , 1.5 5, 1 SP2 1. 3, , 5.5 2,9 58 Avg 51.6 Extraction and preparation of trna was as described in Materials and Methods. Activity was determined by performing the amino acid attachment assay with '4C-L-histidine as the amino acid substrate at a concentration of 2 x 1- IM (1,Ci/ umole). The values shown are averages of four separate determinations from different cultures. McGINNIS AND WILLIAMS repressible by the cognate amino acid, histidine, in the hisr mutant. However, the slight decrease in rate of synthesis of this enzyme in the mutant strain after the addition of histidine was a consistent observation in this study (Fig. 3). Quite convincingly, the data shown in Fig. 3 show a repression of synthesis of histidyl-trna synthetase by histidine for the hisr+ strain. However, previous studies in our laboratory suggested that some changes in synthetase level appeared to be related to the cell densities of the culture (unpublished data). Therefore, experiments similar to those shown in Fig. 3 were performed over a range of starting inoculum sizes and final cell densities for histidine-restricted cultures. These results showed that the response of the rate of synthesis of histidyl-trna synthetase of both strains was unrelated to the cell density of the cultures. Rate of de novo synthesis of histidyltrna synthetase during histidine-restricted and -unrestricted growth. In view of the results (Fig. 3) showing an apparent small degree of repression of histidyl-trna synthetase formation in the mutant strain during unrestricted growth, efforts were made to determine the magnitude of this repressive response by an alternative procedure. For these experiments, use was made of deuterium oxide and water media to create a density difference between molecules of cells grown in histidinerestricted and -unrestricted media. Both strains were grown in deuterium oxide medium containing limiting histidine for two generations, chilled rapidly, washed twice with minimal medium (water), and then resuspended in prewarmed water medium supplemented with excess histidine (1 jg/ml). Figures 4 and 5 present the fractionation, by CsCl centrifugation, of histidyl-trna synthetase of the hisr+ and hisr mutant strains, respectively, into heavy (preexisting) and light (newly synthesized) bands in samples taken at zero time (A), 5% mass increase (B), and 1% mass increase (C) after the density shift. Tabulations of the results shown in Fig. 4 and 5 are presented in Table 2. For the hisr+ strain, the results shown in Fig. 4B and Table 2 indicate that no light (newly synthesized) histidyl-trna synthetase appeared in the culture until after a 5% mass increase in unrestricted medium. Thus, synthesis of this en K N. 28 / 2 24 w U>' S Uremtricted 2 Growth 16 _-+/ t - 8 J. BACTERIOL..4.8.a2.16 BACTERIAL PROTEIN (MG/ML) FIG. 3. Effect of histidine-restricted and -unrestricted growth on the differential rate of formation of histidyl-trna synthetase in the hisr+ (SPI) and the hisr mutant (SP2) strains. The cells were initially grown in unrestricted medium, harvested, washed twice with minimal medium, and transferred to histidine-restricted medium. Then the cells were grown in minimal medium containing limiting histidine (.5 plg of glycyl-dl-histidine/ml). At the time indicated by the arrows, both strains were transferred to histidine-restricted and -unrestricted media. The activity was determined for histidyltrna synthetase of both the hisr+ () and hisr () strains. The results are plotted as enzyme units per milliliter of culture as a function of total protein per milliliter of culture.

5 VOL , 1972 CONTROL OF HISTIDYL-tRNA SYNTHETASE 59 icant repressive effect on the rate of formation 12 - A of histidyl-trna synthetase of the hisr mutant strain. IP=1.33 A plot of the data tabulated in Table 2 is z 6 t \, _ presented in Fig. 6, and shows the differential _ \ rate of formation of histidyl-trna synthetase as determined from the rate of appearance of I- v <this enzyme in the cultures of the hisr+ and C hisr mutant strains. These results support the wi2- B previous data (Fig. 3) showing that, upon the restoration of excess histidine to the derew pressed culture, the synthesis of histidyl-trna I-I synthetase is repressed in the hisr+ strain Z_ (Fig. 6). Conversely, the hisr mutant exhibited nonrepressible synthesis of this enzyme in the z presence of excess histidine, as revealed by c measurement of the rate of de novo synthesis I2 - (Fig. 6). The latter results (Fig. 4-6, Table 2) also indicate that the repression of synthesis of en this synthetase in the wild-type strain involved an actual cessation of de novo synthesis 6_ of this protein molecule. L f i DISCUSSION o The data presented in this paper clearly FRACTION NUMBER FIG. 4. CsCI banding of light and heavy histidyl A trna synthetase units during restricted and unrestricted growth of the hisr+ strain, SPJ. Cells were P=1.33 grown for two generations in histidine-restricted z /\ medium. The first sample (heavy enzyme, A) was E 6 taken at the end of the second generation and 1 Ag, of D L-histidine/ml was added to the cultures. Addi- tional sampling was done at 5% mass increase (B) _ and 1% mass increase (C). Activity of the fractions 'n C is shown as counts per minute of 14C-L-histidine w9 12- B attached to trna under the standard assay condi- I tions. The results shown in this figure are analyzed z further in Table 2. The bottom of the gradient is to n the left of the figure; P indicates the density (g/cc) z< 6 of the molecules in the gradient. a P.1.3 zyme was repressed for,at least,one-half of a a I- ' ' generation of growth after the shift from histi- c) 12 -C dine-restricted to -unrestricted medium (Fig. ' 4B, Table 2). On the other hand, the ratio of heavy to light synthetase units present in cultures of the hisr mutant at the 5% mass in- 6 crease sample indicate that the derepressed (unrestricted) rate of formation of this synthe-, tase was maintained even in the presence of excess histidine (Fig. 5B, Table 2). After one FRACTION NUMBER mass doubling, the light (newly synthesized) FIG. 5. CsCl banding of light and heavy histidylenzyme units present in the hisr mutant cultrna synthetase units during restricted and unreture were approximately twice the predicted stricted growth of the hisr mutant, SP2. The experiamount (Fig. 5C, Table 2). It is clear from the ment was performed as described for Fig. 4. The latter results that the shift from histidine-re- data are plotted as in Fig. 4 and are tabulated in stricted to -unrestricted medium had no signif- Table 2.

6 51 McGINNIS AND WILLIAMS J. BACTERIOL. TABLE 2. Rate of de novo synthesis of histidyl-trna synthetase of strains SP1 and SP2, a hisr mutant, during histidine-restricted and -unrestricted growth as revealed by deuterium labelinga Time Pro- Heavy L after Protein Specific tein enzym Lightd en- Total, enzyme Heavy' en- Light en- Sample den- in activity on on gra- zyme on in culture zyme in zyme in sity culture of gra- dient gradient (units/ml) culture culture shift (mg/ml) extractb dient (units) (units) (units/ml) (units/ml) (min) (mg) Spi A x 1' x 1' 5.99 x 13. B x 1' x lo' 3.85 x 13. C x x x x x 13 SP2 A x x x 13. B x x x x x 13 C x x x x x 13 a Both cultures were grown for many generations in deuterium oxide medium supplemented with excess amounts of all required amino acids. Cells were chilled rapidly, washed twice with deuterium oxide minimal medium, and resuspended in fresh deuterium oxide supplemented with limiting histidine (.5 gg of glycyl- DL-histidine/ml). After two generations of growth, the cells of both strains were washed twice with water minimal medium and shifted to water medium containing 1 jig of L-histidine/ml. Samples were taken at zero time before shift and at 5 and 1% mass increases after the shift to histidine-unrestricted water medium. A portion of each extract was analyzed by CsCl centrifugation in the standard way as shown in Fig. 4 and 5. b Expressed as units per milligram of protein. c Obtained by summing the total heavy enzyme units in the CsCl gradient. d Obtained by summing the total light enzyme units in the CsCl gradients. e Calculated by multiplying the specific activity of each extract by the amount of protein (mg/ml) in the culture at the time of sampling. ' The amounts of heavy and light enzyme per milliliter of culture were calculated from the value for the total amount of enzyme per milliliter of culture by assuming that the relative amounts of heavy and light enzyme were the same as displayed on the CsCl gradients of Fig. 4 and 5. I 1 show that the synthesis of histidyl-trna syno thetase of S. typhimurium is regulated by a x 14 mechanism resembling repression, as has been _j reported for this synthetase in strains of E. coli g 12 (12). The data further suggest that trnahi8 is z involved in the repression of histidyl-trna 1o- synthetase formation. w Histidine restriction of both the hisr+ and U) 8-_ / /o hisr mutant strains caused a derepression of the rate of formation of histidyl-trna synthe- +Histidine tase. This derepressive response to histidine en 6- restriction is specific for histidine, readily de- < / ^/ tected, and quite reproducible. In most experi- _ /ments, a higher level of histidyl-trna synthe- 4-. _j / ^/tase was observed for the hisr mutant than 1 2 _ / /tj ~ that of the wild-type strain during histidine- U) // + Histidine restricted and -unrestricted growth. The signif- I //, icance of this observation is not understood, o.os 6.7 and we plan experiments to examine this ques- BACTERIAL PROTEIN (MG/ ML) FIG. 6. Differential de novo rate of formation of liter of culture taken from Table 2 as a function of histidyl-trna synthetase of strains SPI and SP2 as total protein per milliliter of culture for the hisr+ determined by density labeling. The results in this () and hisr mutant (A) strains. The deuterium oxide figure are calculated from the rates of synthesis histidine-restricted to water histidine-unrestricted measured in Fig. 4 and 5 and listed in Table 2. The shift occurred at the time indicated by the arrow for data are expressed as total enzyme units per milli- each culture.

7 VOL. 19, 1972 tion further. Restoration of histidine to the derepressed hisr+ culture caused an immediate cessation of synthesis of the synthetase for, at least, one-half mass doubling, despite the resumption of the unrestricted rate of growth. In contrast, addition of histidine to the derepressed hisr mutant culture had little, if any, repressive effect on the rate of formation of histidyl-trna synthetase. In fact, the hisr mutant continued to synthesize this synthetase at the derepressed rate despite the presence of histidine and even though the unrestricted rate of growth was resumed. The density labeling data provide evidence that the de novo rate of formation of histidyltrna synthetase was immediately repressed by histidine in the hisr+ strain. More interestingly, these experiments provided data which clearly indicate that histidine did not cause a repression of the rate of de novo synthesis of this enzyme in the hisr mutant. Furthermore, the structural genes for histidyl-trna synthetase and trnahis lie on the S. typhimurium chromosome at some distance from the histidine operon. There is evidence for two hisr genes that are adjacent and coding for identical trna species as revealed by studying the presently available hisr mutants (P. Hartman, personal communications). This raises the question of whether the regulatory elements involved in the control of synthesis of the biosynthetic enzymes and the synthetase are distinct biochemical units. This point is made even more important by the apparent parallel repression of synthesis of the two classes of histidine enzymes, as observed in the present study. However, despite the obvious similarity in response to histidine restriction and dependence on trnahi it is not clear whether trnah1s charged with histidine or some derivative thereof is the co-repressor for both classes of enzymes. To provide answers to these questions, we plan studies of synthetase regulation in different hisr mutants (i.e., different amounts of trnahis) and mutants with altered regulation of the histidine biosynthetic enzymes. CONTROL OF HISTIDYL-tRNA SYNTHETASE 511 ACKNOWLEDGMENTS This work was supported by Public Health Service research grant GM from the National Institute of General Medical Sciences and by American Cancer Society grant p574. Luther S. Williams is a public Health Service Career Development awardee (K4-GM ). We are grateful to Mark Levinthal, Molecular Biology Laboratory, National Institutes of Health, for providing the two parental strains of S. typhimurium used in this study and for many helpful suggestions. We also express appreciation to H. E. Umbarger of Purdue University for helpful discussions throughout this investigation. LITERATURE CITED 1. Ames, B. N., B. Garry, and L. A. Herzenberg The genetic control of enzymes of histidine biosynthesis in Salmonella typhimurium. J. Gen. Microbiol. 22: Chrispeels, M. M., R. F. Boyd, L. S. Williams, and F. C. Neidhardt Modification of valyl-trna synthetase by bacteriophage in Escherichia coli. J. Mol. Biol. 31: Fraenkel, D. G., and F. C. Neidhardt Use of chloramphenicol to study control of RNA synthesis in bacteria. Biochim. Biophys. Acta 53: Gorini, L., and H. Kaufman Selecting bacterial mutants by the penicillin method. Science 131: Lowry,. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: McGinnis, E., and L. S. Williams Regulation of synthesis of the aminoacyl-transfer ribonucleic acid synthetases for the branched-chain amino acids of Escherichia coli. J. Bacteriol. 18: Nass, G., and F. C. Neidhardt Regulation of formation of aminoacyl-ribonucleic acid synthetase in Escherichia coli. Biochim. Biophys. Acta 134: Roth, J., and B. N. Ames Histidine regulatory mutant having altered histidyl-trna synthetase. J. Mol. Biol. 22: Roth, J. R., D. Silbert, G. Fink, M. J. Voll, D. Ant6n, P. E. Hartman, and B. N. Ames Transfer-RNA and the control of the histidine operon. Cold Spring Harbor Symp. Quant. Biol. 31: Silbert, D., G. Fink, and B. N. Ames Histidine regulatory mutants in Salmonella typhimurium. III. A class of regulatory mutants deficient in trna for histidine. J. Mol. Biol. 22: Williams, L. S., and M. Freundlich Control of isoleucine, valine and leucine biosynthesis. VII. Role of valine transfer RNA in repression. Biochim. Biophys. Acta 186: Williams, L. S., and F. C. Neidhardt Synthesis and inactivation of aminoacyl-trna synthetases during growth of Escherichia coli. J. Mol. Biol. 43: