Histidine Regulation in Salmonella typhimurium

Mutations in any of six genes of Salmonella iyphimurium lead to constitutivity of the histidine operon. Four of these genes, hisR, hid, hisW, and hisT, are believed to be involved with the production of histidine tRNA. We show that the tRNAHiB isolated from each of these mutants is aminoacylated normally, indicating that the constitutivity of these mutants cannot be ascribed to a reduced ability of their histidine tRNA to be charged. HisT mutants are known to have a structurally altered tRNAHiS. These mutants are apparently constitutive because of failure of the altered tRNA to interact properly with the repression mechanism. The binding of charged tRNAniS from hisT mutants to the histidyl-tRNA synthetase, one candidate for the corepressor of the histidine operon, has been tested by filter binding. Under several different assay conditions the binding of histidyl-tRNAHiS from the wild type and from hisT1504 is the same within experimental error. An assay for the enzymatic deacylation of histidyl-tRNA”‘” was developed to check the binding constants obtained by the filter binding method. The synthetase has surprisingly low K, values for the substrates of the reverse reaction; 45 pM for AMP and 15 PM for pyrophosphate. The K, for charged tRNAniS, 40 nM, is the same as the dissociation constant found for charged tRNA”‘” by filter binding and also the same as the K, for uncharged tRNAHi” in the aminoacylation reaction. The K, of the enzyme for tRNAHiS is shown to increase as the histidine concentration is increased, over a range of histidine concentrations that are found physiologically. It is shown that an increase in pyrophosphate concentration increases the inhibitory effect of AMP an synthetase activity, and it is suggested that this has physiological meaning.


Mutations
in any of six genes of Salmonella iyphimurium lead to constitutivity of the histidine operon.
Four of these genes, hisR, hid, hisW, and hisT, are believed to be involved with the production of histidine tRNA. We show that the tRNAHiB isolated from each of these mutants is aminoacylated normally, indicating that the constitutivity of these mutants cannot be ascribed to a reduced ability of their histidine tRNA to be charged. HisT mutants are known to have a structurally altered tRNAHiS.
These mutants are apparently constitutive because of failure of the altered tRNA to interact properly with the repression mechanism.
The binding of charged tRNAniS from hisT mutants to the histidyl-tRNA synthetase, one candidate for the corepressor of the histidine operon, has been tested by filter binding.
Under several different assay conditions the binding of histidyl-tRNAHiS from the wild type and from hisT1504 is the same within experimental error.
An assay for the enzymatic deacylation of histidyl-tRNA"'" was developed to check the binding constants obtained by the filter binding method. The synthetase has surprisingly low K, values for the substrates of the reverse reaction; 45 pM for AMP and 15 PM for pyrophosphate.
The K, for charged tRNAniS, 40 nM, is the same as the dissociation constant found for charged tRNA"'" by filter binding and also the same as the K, for uncharged tRNAHi" in the aminoacylation reaction.
The K, of the enzyme for tRNAHiS is shown to increase as the histidine concentration is increased, over a range of histidine concentrations that are found physiologically.
It is shown that an increase in pyrophosphate concentration increases the inhibitory effect of AMP an synthetase activity, and it is suggested that this has physiological meaning.
* The 13th paper of this series is ltefercnce 1. The histidine operon of Suln~on~lla ~!/p/Cr ctriunt is compo+~ I of the nine genes which code for the eiizgmes of lristidinr biosynthesis.
These genes occupy c*ontiguous positions on thtx &zlmo~zella chromosome, and their activity is controlled as a unit, in response to t.he need of the cell for histidine hiosynthesi,\.
To unravel the mechanism of coiitrol of the activity of thesr~ genes, a large number of constitutive mutants have been isoiatr~l. The constitutive mutations map in sis different chromosorrlal locations (2)(3)(4)(5)(6).
The mutations of one set map at. the begiruliirl: of the operon and are cis dominant, suggesting t,lrat they are operator mutations (7,8). The remaining loci, AisR, hisS, hisT, hinl-, and hisW, are all recessive (7). Each of these I'+ maining loci has been implicated iu the productioil of tRK.\T'iS or in its charging.
Hid codes for the Iiistidyl-tR?;.\ syntheta~e (3,9), hisR appears to be a structural gene for histidine tRS.\ (lo-la), and hisT codes for an enzyme which converts uritlitw to pseudouridine in the anticodon loop of the histidiite tRNA (13) HisC and lrisll~ may also be iuvolved iit tRSA nrat,uratiorl, :W both mutant types appear t)o have reduced levels of several species of tRNh (12). In this paper we examine the interaction of the histidyl-t,ltX.\ synthetase with histidine tRNX isolated from the constitmivrt mutants.
These studies are divided into two parts. In tht> first part, both the K, and r,,,,, of t,RNAHiS isolated froril t,ht: mutant strains were determined in the :llninoa.cSrl:ltioIl reartiotr. These studies serve as a probe for ljossible differeirces in tRN.\ structure, and as a test of the possibility th:rt the constitutivit~ of these strains might result from reduced charging of an altered histidine tRNA. The second lxnt of t,lle ~)aper es;rnlirrcs t IIC possibility that the hi&idyllt,RT.\ synthetase, in complex I\-itli charged histidine tRNA, might be the repressor for the operoil (14). 111 these experiments the binding to the enzyme of chargt~l histidine tRN.4 from hisT mutarns is compared with that frorrl the wild type.
Histidine tRKi\ is known to be present in normal quantities in hisT mutants, alrd to be charged to the iiormal estent (11, 12), making it likely that these mutants are derepressetl because of failure of the charged tRNIZ to interact properly n-it,11 the repression mechanism.
If biuding of charged histidine tRS.1 to the synthetase is required for repression, it might be esprc%ect that charged tRX:L from hisT mutants \vould bind less t,ighti>to the enzyme than the wild type tHNlZ.
The lrinding studies with charged tRNAHis were done using the filter bintliilg The radioactive histidine was stored at -20", and before use an aliquot was evaporated to dryness and dissolved in an equal volume of water.
The crystalline sodium salt of ATP, unlabeled L-histidine, and bovine serum albumin, type V, were obtained from Sigma. AMP was a product of Schwartz BioResearch, and sodium pyrophosphate was from Mallinckrodt. Homogeneously pure histidyl-tRNA synthetase (specific activity 5000 units per mg) was prepared as described previously (16). Glass fiber filters (type A, 1 inch in diameter) were purchased from Gelman Instrument Company. Determination of K, and V,,,,, for tRNAHi" in Amirwacylation Reaction-In a final volume of 0.25 ml the reaction mixture contained 8 rniM MgC12, 4 mM AT!?, 15 PM [3H]histidine, and 0.1 M sodium cacodylate at pH 7.5. The concentration of tRNAHi8 was varied from about 20 nM to 200 nM. Tubes were incubated previously for 2 min and enzyme added to start the reaction. When assays were run at 37", histidyl-tRNA synthetase was usually used at a final concentration of 6.1 ng per ml; when assays were run at 20" the enzyme concentration was usually 15 ng per ml.
Samples of 75 ~1 were taken at 1, 2, and 3 min, the reaction stopped by the addition of 3 ml of cold 10% trichloroacetic acid, and the resulting suspension chilled in ice. The precipitate was collected on a glass fiber disc and washed four times with 5 ml of lOy0 trichloroacetic acid, four times with 5 ml of 957, ethanol, and twice with 5 ml of ether. The air-dried filters were counted in 10 ml of a mixture of toluene, 2,5-diphenyloxazole @PO), and 1,4-bis-[2-(5-phenyloxazolyl)]benzene (POPOP) (Spectrafluor, Amersham/Searle) in a Nuclear-Chicago Mark I liquid scintillation counter at an efficiency of 30%. Preparation of Histidyl-tRNAHi"-The tRNAHi8 is aminoacylated using the reaction conditions described above, except that a large excess of enzyme is used to insure that the reaction goes rapidly to completion. The reaction is stopped by the addition of 13 volumes of 67% ethanol at -2O", which contains 10 nl M MgClz and 10 mM sodium acetate, pH 4.5 (the large volume of ethanol is necessary to keep the ATP in solution). The precipitate is collected by filtration through Millipore filters, type HA, 0.45 ~1 (Millipore Filt.er Corporation, New Bedford, Mass.), and washed with 20 ml of the buffered alcohol.
Tip to 50 AZ6,, units of tRNA are collected on a single RIillipore filter. The filters are air dried, and the tRNA eluted into a buffer containing 1 mM MgC12 and 1 rnM sodium acetate, pH 4.5, by shaking in the cold for 15 min.
It was found convenient to perform the elutioll in a glass scintillation vial, usually with 1 ml of buffer. A slight variation of this procedure was used to prepare charged tRNAHiS for the binding reaction. The tRNA was charged to completion as described above, then the reaction was quenched by the addition of 0.25 volume of ice-cold 1 M acetic acid followed by 2.5 volumes of ice-cold water.
After the column was washed with 45 ml of the equilibration buffer, the column was eluted with the same buffer made 1 M in NaCl and 3-ml fractions were collected.
High salt fractions containing radioactivity were pooled and mixed with 2 volumes of ethanol at -18".
After 1 hour at -18", the precipitated nucleic acid was collected by vacuum filtration on several Millipore filt,ers according to the method described above.
Collection of tRNA on membrane filters has proved to be :t convenient method for dealing with small quantities of tRNh. This method has been used with total amounts as small as 0.1 A260 unit (5 pg) and concentrations as low as 0.01 A~Q unit pel ml.
A number of different filter types have been screened to select that most suited for collection of tRNA samples. The Millipore filter type HA, 0.45 p, proved to be superior to eight other types tested for tRNA collection (171. In using these filters, however, one must not exceed an alcohol concentration of about 75%, as higher concentrations (95%) partially dissolx e the filters.
Elution of tRNA from the filters is rapid, and does not appear to be sensitive to the buffer used. The elution is about 95% complete in 5 min, and lOOo/;, complete by 15  added to initiate the reaction. At intervals of 1, 2, and 3 min, 75+1 samples were withdrawn and the reaction quenched by the addition of 1 ml of 5% trichloroacetic acid. The chilled precipitate was then filtered through a glass fiber filter.
In some experiments 0.5 ml of the filtrate was neutralized with 0.1 ml of a NaOH solution 0.1 M in sodium cacody1at.e and counted in Bray's scintillation fluid (18). In other experiments 0.2 ml of the filtrate was pipetted onto glass fiber filters, the filters dried at 120", and then counted in the 2,5-diphenyloxazole-1,4-bis-[2-(5.phenyloxazolyl)] benzene-toluene solution described above.
Counting was done in a Nuclear Chicago Mark I liquid scintillation counter. In Bray's solution efficiencies of 15'i/, for 3H and 80% for 14C were obtained.
Binding Assay-Histidyl-tRN,L\ synthetase and tRNX"" charged with histidine were incubated together in 120 ~1 of reaction mix of composition described in the figures and tables. At the end of the incubation period, a lOO+l sample was withdrawn and filtered through a Schleicher and Schuell B-6 nitrocellulose filter, previously moistened in water.
The filter was then washed with 0.6 ml of the buffer used in the assay, dried for 10 min at llO", and radioactivity determined using the 2,5-di- and C, LT-2 (wild type).

Kinetic
Analysis of tRNA in Amiruwylation Reaction--Both the K, and I',,, were determined for histidine tRNA isolated from the wild type and from the regulatory mutants, hisR, hidI, hisW, hisT, and hisO. The tRNA from his0 served as a derepressed control, since his0 is known to be an operator region (7,8,19), and therefore not involved with tRNA production. Typical double reciprocal plots of the kinetic data are presented in Fig. 1. Table I presents the K, and V,,, values determined. As can be seen, the parameters determined for tRNA isolated from the mutant strains do not differ significantly from those determined for tRNA isolated from the wild type. The sensitivity of this probe of tRNA structure is limited by the variation found between different preparations of the wild type tRNA. This is particularly noticeable for the first preparation listed. This batch of tkNA was that used to determine the published value of 110 no for the tRNAHis K, (16), and that value is confirmed here. However, examination of the data in Table I reveals a K, of about 40 no to be more representative. It is perhaps significant that all of the tRNA preparations assayed have been prepared within the last year except various strains Assays were performed as described in "Materials and Methods." Values shown in brackets were determined at 20"; all other values were determined at 37". Independent preparations of tRNA are denoted by repetition of the strain designation. The "standard" preparation is made by phenol extraction of the cells followed by chromatography on DEAE-cellulose, as described in Reference 11. The "Silbert" method is essentially the same, except LiCl replaces NaCl in the buffers (10). The "thiosulfate" preparations are the same as the "standard," except 2 mM sodium thiosulfate was present in all buffers to protect sulfhydryl groups in the tRNA (11 P21 that giving the higher K,,, value, which is 5 years old. It is possible that some modification has been produced upon prolonged storage which has altered the affinity of that tRNA for the synthetase. The reason for the variation in tRNA Km for the same preparation assayed at different times is not known. It is not due to aging of the enzyme upon storage, as freshly prepared enzyme gives essentially the same K, (perhaps 25% higher) as enzyme which has been stored over a year at -20" in 50% glycerol. Also ruled out is a possible slow change in the enzyme during the variable interval between dilution for assay and use in the assay. When the enzyme is left in ice for 2) hours after dilution (the maximum time interval encountered), the K,,, for tRNAHi* remains unchanged.
Among the tRNA's analyzed was that from the cold-sensitive hisW mutant, JL250. To determine if the immediate cessation of growth displayed by this mutant when shifted to 20" was due to a cold-sensitive tRNAHiS, the K, for the JL250 tRNAHiS was also determined at this lower temperature (Table  I, values in brackets). No dramatic difference in K,,, or Vma, was found for the tRNAHiB of the mutant relative to that from a number of other Salmonella strains which were assayed as controls.
Measurements of the K, for tRNAHis discussed above were made at a histidine concentration of 15 /AM, the concentration of the internal histidine pool of Salmonella growing on minimal salts-glucose medium (20). As shown in Fig. 2 We are not sure whether this variation of tRNA K, with histidine concentration has any physiological significance, since the in vivo concentrations of the synthetase and uncharged tRNAHi8 (1.5 PM and 0.23 MM, respectively (12, 16, li')), are much higher than the Km of the enzyme for the tRNA (~0.04 PM).
Binding of Charged Histidine tRNA to Histidyl-tRNA Synthetase-HisT mutants produce a structurally altered histidine tRNA (11, 13). However, the mutant tRNA is present in the cell in wild type quantity (11, 12) and is charged normally (12 ,  Table 'I). -Apparently hisT mutants are derepressed through failure of the charged histidine tRNA of h&T mutants to interact properly with the repression mechanism.
If the histidyl-tRNA synthetase is the corepressor for the histidine operon (14), one might expect it to bind charged histidine tRNA from hisT mutants less strongly than the charged tRNA from the wild type. This prediction was tested using the filter binding method for measuring the affinity of tRNA for the enzyme. The method used was essentially that of Yarus and Berg (15), except that the reaction conditions were modified to more closely approximate the physiological conditions under which repression of the histidine operon has been studied in S. typhimurium. Accordingly, the incubations were done at pH 7.0, rather than pH 5.5, and at temperatures of 25" and 37", rather than 17". In addition, the data of Yarus and Berg (21) indicate that formation of synthetase-isoleucyl-AMP complex occurs faster than dissociation of charged tRNA"" from the enqme. If the same is true for the histidyl-tRNA synthetase, the form of the synthetase expected to predominate in vivo during conditions of repression is the synthetase-histidyl-AMP complex. Thus, we also studied the binding of charged tRNAHiS from the wild type and from h.isTlBOA to the synthetase-histidyl-AMP complex as well as sodium phosphate buffer, A--A. Samples were then filtered and counted as described under "Materials and Methods." Binding was also measured using synthetase which had been incubated for 10 min in 120 ~1 of 50 mM cacodylate buffer containing 10 mM MgC12, 15 PM unlabeled histidine, and 4 mM ATP. The apparent lesser binding of charged tRNAHim to the histidyl-AMP-synthetsse complex below pH 6 is probably due to denaturation of the synthetase at low pH during this preliminary incubation. After preincubation tRNA was added to give a final reaction volume of 130 ~1 (O-O ). All assays were at room temperature and were as described under "Materials and Methods." their binding to the synthetase alone. When required, the histidyl-AMP-synthetase complex was generated by preliminary incubation of the enzyme with 15 pM histidine and 4 mM ATP for 10 to 25 min.
Several buffers were tested for their efficacy in the binding reaction at pH 7.0 (Table II).
Sodium cacodylate was selected for use in subsequent assays as it permitted a high level of binding while maintaining a low blank. As shown in the table, the amount of synthetase-tRNA complex trapped on the filter in the presence of cacodylate is 6 times that trapped in the presence of phosphate buffer. Fig. 3 shows that cacodylate buffer allows detection of considerably more complex than phosphate buffer at all pH values tested in the pH range of 5.5 to 7.5. Significant! y, the synthetase-histidyl-AMP complex binds more charged tRNAHi8 in cacodylate buffer between pH 6.0 and 7.5 than the synthetase alone.
The time of incubation with charged tRNAHiB has negligible effect on complex detection. Within experimental error the same level of complex was detected with incubation times ranging from 20 s to 5 min. There was also little effect of temperature between 0" and 37". Essentially the same level of binding was found at 0" and 15', and decreases of only about 12 and 16% were noted at 25" and 37", respectively.
The effects of incubation time and temperature were the same whether the charged tRNXHis was bound to the synthetase alone or to the histidyl-hMP-synthetase complex. However, in all cases, at pH 7.0 the histidyl-AMP-synthetase complex bound 4 to 5 times more charged tRNAHi8 than did the synthetase alone.
Binding to the synthetase alone of histidyl-tRNAHis from the wild type and from hisT1504 was measured at room temperature (24"), and binding to the histidyl-AMP-synthetase complex was measured at both 24" and 37". The dissociation constants obtained are presented in Table III, and a typical experiment is shown in Fig. 4. Within the range of variation the results for wild type and hisT1504 tRNh are the same. The results also indicate that the binding of charged tRNAHiS from both strains to the synthetase-histidyl-AMP complex is less tight than binding to the free enzyme. This effect parallels the increase in tRNAHi" K, with increasing histidine concentration noted in the aminoacylation reaction (Fig. 2). Binding of wild type and hisTl604 histidyl-tRNAHi8 to the histidyl-tRNA synthetase in the presence of ATP and histidine at 37". One hundred ten picomoles of synthetase were preincubated for 10 to 25 min at 24' in 15 fiM unlabeled histidine, 4 mM ATP, 10 mM MgC12, and 50 mM sodium cacodylate buffer, pH 7.0. The temperature was then changed to 37", and various amounts of charged tRNAHi# from wild type or hisTl604 were added. After 15 s, a sample was filtered and counted as described under "Materials and Methods." The total reaction volume was 130 ~1.
are bound without the addition and with the addition of ATP and histidine, respectively. Our synthktase preparation is clearly not fully active. Presumably the increased binding in the presence of ATP and histidine is due to the activation of some of the enzyme molecules by these substrates, although this has not been tested. For these experiments we employed the highly purified, homogeneous material described by De Lorenzo and Ames (161. Our enzyme has a specific activity similar to the activity of freshly purified enzyme as judged by its ability to charge tRNAHi8. The reasons for the low binding activity and the variability in the determination of dissociation constants are currently under investigation. and also with sufficient speed that nonenzymatic deacylation of the charged tRNA is inconsequential.
Properties of Deacylation Assay-The reaction rate is linear with time and with enzyme concentration in the range used. In addition, little or no nonenzymatic deacylation occurs during the time of the assay. The reaction is almost totally dependent upon the presence of AMP and pyrophosphate; in the absence of these substrates the rate of enzymatic deacylation is reduced 1000.fold.
Substrate K, Values-The K, for pyrophosphate is about 15 pM.
Data from a typical experiment are plotted in Fig. 5. AMP has a K, near 45 pM (Fig. 6). The low value for the AMP K, is somewhat surprising, since it is only one-third the K, for ATP in the forward reaction (16).
The K, for histidyl-tRNAHiS is about 40 no (Fig. 7). This value is within experimental error of that for uncharged tRNL4HiS in the forward reaction (Table I), and agrees closely with the binding constant found by the filter binding procedure. nism of the filter binding assay is not known.
In particular, it is quest,ionable whether the conformation which the enzyme assumes upon binding to the filter is representative of its native conformation; and therefore, whether the binding constants determined by this method truly represent the affinity between native tRNA and enzyme. Thus it is desirable to have an independent method of measuring the binding constant to serve as a check upon the filter binding procedure.
We have exploited the reversal of the aminoacylation reaction for this purpose. The enzymatic deacylation of histidyl-tRNA can be conducted at a temperature and pH likely to represent those within the cell, DISCUSSION In this paper we examined the interaction of the histidyl-tRNA synthetase with histidine tRNA isolated from those constitutive mutants possibly defective in tRNA production.
Since several of these strains are thought to have an altered tRNAHis, it was considered that constitutivity might result from failure of these tRNA's to be charged normally. Accordingly, the Km and V,,, for the tRNA in the aminoacylation reaction were determined.
Failure to find any kinetic difference between tRNAHis isolated from the mutant and wild type strains suggests the tRNAHiS in these strains should be charged to the normal extent in viva. This prediction has recently been verified (12), requiring alternate hypotheses to be considered for the cause of constitutivity.
In the case of hisR, constitutivity likely arises from a decrease in the total level of histidine tRNA in the cell (l&12).
A lowering of the histidine tRNA content of his?7 and hisW strains may also be responsible for the constitutivity of these mutants (12). HisT mutants, however, appear to have both the normal content of histidine tRNA, and normal charging of that tRNA within the cell (11, 12). At the same time, hisT mutants are known to produce a structurally altered histidine tRNh (11, 13). It would appear, therefore, that constitutivity of hisT mutants is caused by a failure of the charged histidine tRNA in h&T mutants to interact properly with the repression mechanism.
One possibility which we have been considering is that the histidyl-tRNA synthetase, in complex with charged histidine tRN.4, is the repressor for the histidine operon (14). If such were the case, one might expect charged histidine ttlN;\ from the wild type to bind to the enzyme, but that the tRNA from h,isT mutants would not. However, the data presented here show that caharged tRNA"'" from 1risT mutants binds with the same :If&rity as the wild type. This finding is consonant with the results of the :~nliIloacyl:~tioI~ reaction, in which uncharged tlIN,\"'" from tile wild type :d from hisT1504 yielded the same K,,. .\lthough these results do not support the hypothesis that the syllthetnse is the corepressor of the histidine operon, neither do they rule it out. '1'11~ altered structure of charged tRNAHi8 from l&T mutant-; might cause the synthetase to function in-effic+ntly as it (*orepressor while still causing no significant change iI1 the st,rength of tRN,1 binding to t,he synthetase. l~iochemical :III~ genetic studies are in progress to test more conclusively the l~o~sibility that the histidyl-t1iY.L sytrthetase is the c*oregulator of the Ilistidiilo operoil. L\n assay of the enzymatic tleacylation of charged tRN:\ was develoI)ed to check the validity of the filter binding assay. The dear)-lation reaction n>:Ly prove valuable for the analysis of enzyme-tRNA illteractiolls it) systenrs where filter binding does Ilot work, particularly when l)hysiological conditions are desired. The reaction may also be useful for kinetic analysis of the reaction mechanism.
Of particular interest is the low I<, (45 pear) for :0Il' in the clescylation reaction.
This Zi,, is about a-fold lower than that for hT1' in the forward react,ion. It has been shown that the R,, for .Ln4I' is strongly dependent upon the concentration of pyrophospliate in the reaction mixture (17). The K,,, for AMI' doubles when the pyropltosphate concentration is reduced from one which saturates t,he enzyme (1 mbi) to its I<, value (15 pal). The histidyl-tRN. 2 synthetase leas previously been shown to be subject to energy charge control (inhibition by AMP and ADI') in the absence of pyropltosphate (22). Since the presence of pgrophosphate increases the binding of -%\IP, energy charge control sltould be stronger when pyrephosphate is present, and this may well have physiological meaning. Xo data have been found concerning the in viva concentration of pyrophosphnte in bacteria, but pyrophosphate concentrations as high RS a millimolar have been recently report.ed for rat and guinea pig liver (23).
The filter binding assay and the deacylation assay give the same binding const,ant for charged lristidine tRN:Z as is found for uncharged tRh :\ T His iu the aminoacylation react,ion. 'She similarity in binding constants could not hvc llern predicted u priori: although charged and mkcharged tRNA are similar ill gross structure, several workers 11ave found that charging ol tlCN.1 leads to a change in its conformation (24-27). Such cli:ulges in (Bollformation could lower the affinity of the charged IGochem.
SW. /.rll. 9, 327 -tRNA for the synthetase. The strong binding which is observed between the synthetase and histidine tRNA has two interesting implications.
First, even if the synthetase-charged tRN.4 complex is not directly involved in repression, the ability of the synthetase to complex a large portion of the charged tRNA in the cell must be considered when describing the mechanism of control of the histidine operon.
Second, the strong binding of charged tRNh suggests that it is a good inhibitor of the aminoacylation reaction.
We have found this to be the case.] Since approximately 800/ of the histidine tRNA in the cell is in the charged state (12), only a fraction of the synthetase can be espetted to be active in vivo. Suc11 product inhibition might serve to regulate the rate of the aminoacylation reaction.