Vol. 139, No. 1

JOURNAL OF BACRziOLOGY, July 1979, p. 176-184 0021-9193/79/07-0176/09$02.00/0

Regulation of the Biosynthesis of Aminoacyl-Transfer Ribonucleic Acid Synthetases and of Transfer Ribonucleic Acid in Escherichia coli VI. Mutants with Increased Levels of Glutaminyl-Transfer Ribonucleic Acid Synthetase and of Glutamine Transfer Ribonucleic Acid ALICE CHEUNG, SUSAN MORGAN, K. BROOKS LOW, AND DIETER SOLL Department of Molecular Biophysics and Biochemistry, and Department of Therapeutic Radiology, Yale University, New Haven, Connecticut 06520 Received for publication 18 January 1979

Spontaneous revertants of a temperature-sensitive Escherichia coli strain

bearing a thennolabile glutaminyl-transfer ribonucleic acid (tRNA) synthetase have been selected for growth at 450C. Among 10 revertants still containing the thermolabile enzyme, 2 interesting strains were found. One strain has a fivefold elevated level of the thermolabile glutaminyl-tRNA synthetase; the genetic locus, glnR, responsible for this effect maps at min 24, far from gInS, the structural gene of the enzyme. In the other strain the levels of tRNAG and several other tRNAs are twice as high as in the parental strain; the locus responsible, glnU, maps at min 59.5 on the E. coli map. tRNA and aminoacyl-tRNA synthetases are portunities for posttranscriptional regulation of

two classes of macromolecules that play key tRNA levels. Detailed infofmation regarding roles in protein synthesis (22, 23). An under- these questions is only slowly forthcoming, parstanding of the factors controlling the levels of ticularly since genetic studies on tRNA, except these macromolecules in the cell is of great those that serve suppression purposes (23a), interest; it may also shed light on the regulation have been very limited. This is partly due to the of processes in which these molecules are known difficulty in isolating strains which are mutated in tRNA genes: on one hand, mutations in single to be involved. Our understanding of the regulation of tRNA copy tRNA genes may be lethal; on the other, biosynthesis is still very limited even in E. coli, the effect of a mutation in one gene of an isoacwhich has been studied most extensively so far ceptor RNA with many gene copies in the chro(20). The number of tRNA genes coding for a mosome may not be significant enough to be particular tRNA species and their chromosomal exhibited phenotypically. We have described earlier the isolation of a locations are determined only for a fraction of the tRNA's (9, 18). Glutamine tRNA (tRNAGl'n) mutation in E. coli which leads to increased represents roughly 2% of unfractionated E. coli production of tRNA1GIn (19). The isolation of tRNA and occurs in two isoacceptor species (27). this mutant was possible because a mutation Physical and genetic studies have shown at least leading to elevated tRNA levels suppressed the two genes for either species situated adjacent to temperature sensitivity of a strain harboring a each other (H. Inokuchi, M. Kodaira, F. Yamao, thermolabile glutaminyl-tRNA synthetase (11). and H. Ozeki, unpublished data). This is proba- Other mutations resulting in the same phenobly not the full complement of tRNAG' genes; type may give rise to strains with increased the glnT locus may represent gene(s) for levels of the defective aminoacyl-tRNA synthetRNA1Gln (19), and the mapping of tRNA genes tase (3, 14, 24). In this study we selected other temperaturein E. coli is not yet completed. Knowledge of the number of tRNA genes and the different kind of resistant revertants from a temper,ture-sensitRNA genes in a transcriptional unit is a prereq- tive glnS strain. Among these strains we found uisite for an understanding of transcriptional two with regulatory mutations: one, glnR inregulation by a single RNA polymerase. In ad- creases the level of the thennolabile glutaminyldition, the complicated enzymatic mechanism of tRNA synthetase and the other, glnU increases tRNA biosynthesis involving many nucleases the level of tRNAGlD and several other tRNA and tRNA modifying enzymes gives ample op- species. 176

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MATERIALS AND METHODS The procedures used in this study were described in the preceding paper (3). Exceptions are listed below. Uniformly labeled ['4C]glutamine (213 mCi/mmol) and [3H]glutamine (21 Ci/mmol) were obtained commercially. All other 14C-labeled amino acids used had a specific activity of 200 to 325 mCi/mmol. [3H]methionine had a specific activity of 10 Ci/mol. Rabbit anti-glutaminyl-tRNA synthetase serum was prepared by A. Korner (11). Bacterial strains. The bacterial strains used in this study are described in Table 1. Revertant strains. Spontaneous temperature-resistant revertants of strain AB4143 were selected on Luria and Burrows (LB) agar at 45°C. Portions (0.1 ml) were removed from a fresh 5-ml LB culture grown at 30°C and plated on LB agar. The colonies which appeared after incubation at 45°C were picked and retested by further replica plating. Reversed-phase chromatography (RPC-5) of tRNA. The column material, trialkylmethylammonium chloride supported on Plaskon, was prepared as described by Pearson et al. (21). Chromatography was performed at 37°C using 0.01 M sodium acetate (pH 4.5)-0.01 M MgCl2-0.01 M 2-mercaptoethanol in all buffers. Columns (0.9 by 81 cm) were equilibrated with 0.45 M NaCl, eluted with a linear gradient (400 ml) from 0.45 to 0.7 M NaCl, and washed with 1.5 M NaCl. A constant flow rate of 1.0 ml/min was achieved by using a Milton Roy MiniPump (150 to 250 lb/in2), and 2-ml fractions were collected. Determination of in vivo levels of glutaminyltRNA. Cells were grown in either 30 or 39°C in minimal medium. The procedure of Morgan et al. (19) was followed by using acetylated dihydroxyboryl-substituted cellulose (17) to separate aminoacyl-tRNA from

177

tRNA. Determination of amino acid pools. The free amino acids were extracted from E. coli as described by Hayashi et al. (8). Cultures (15 ml) were grown in supplemented minimal medium to a density of 3 x 108 cells per ml. The cells were collected by filtration on a membrane filter (Millipore Corp.) and washed once with 15 ml of unsupplemented minimal medium. Free amino acids were released by osmotic shock (5) when 15 ml of cold distilled water was passed through the filter. The filtrate was concentrated by rotary evaporation, and the amino acid concentrations were determined by utilizing an amino acid analyzer. Enzyme assays. Cells were grown at 39°C in minimal medium. Glutamine synthetase. The biosynthetic activity of glutamine synthetase was determined by the phosphate release assay (26). One unit of enzyme is defined as that amount required to catalyze the synthesis of 1 ,.mol of Pi per min. The amount of glutamine synthetase was assayed by the transferase reaction (26). One unit of enzyme activity is defined as that amount required to catalyze the synthesis of 1 umol of gammaglutamylhydroxamate per min. Histidinol phosphate phosphatase. This enzyme was assayed as described by Ely (7). The enzymatic cleavage of histidinol phosphate to histidinol and Pi is followed colorimetrically. One unit of enzyme is defined as the formation of 1 unit of absorbance at 280 nm (A2so) in 15 min.

RESULTS Isolation of temperature-resistant revertants and initial characterization. Spontaneous temperature-resistant revertants of

TABLE 1. List of strains Strain

W3110 AB3441

Genotype

A-

KL476

F8( gar+)/galK2 thi-1 metC56 xyl ara-14 lacYl tfr-5 tsx-57 supE44 As for AB3441, but F- ginSl str-71 As for AB4143, but gInR5

KL477

As for AB4143, but glnU6

KL478 KL479 KL16 KL267

As for KL476, but thyA140 As for KL477, but thyA141 Hfr thi-l rel-l XHfr, as KL16, but cysC43 argA21 deoB3 As for KL477, but cysC43 argA21 F15(thy+)/metBI leu-6 his-i argG6 lacYI or Z4 gal-6 tonA2 xyl- 7 mtl-2 malAU sup55 str-104 recAl thy Ar AF15(thy+)/KL476 F- proA2 purB15 his-4 thi-1 str-35 lacYl, galK12 mtl-I xyl-5 ginSl F- pyrC46purB51, lacZ43 or lacZ13 thi-1, malAI, mtl-2 xyl- 7 str-125 AAs for X7014a, but maL4' and ginSl

AB4143

KL480

F15/KL110 F15/KL476 KL304

X7014a

KL481

Source

Bachmann (1) Korner et al. (11)

Korner et al. (11) Temperature-resistant revertant of AB4143 Temperature-resistant revertant of AB4143 KL476 - Thy- trimethoprimr KL477 Thy- trimethoprimr Low (16) Lloyd et al. (15) KL267 x KL479 Low (16)

-*

Thy+ (Strr)

F15/KL110 x KL478 -. Thy' (Strr) Made similar to KL303 (11) Bachmann

F15/KL476 x X7014a -- Mal+ (Met+)

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strain AB4143 (carrying a thermolabile glutaminyl-tRNA synthetase and unable to grow at about 400C) were isolated on LB agar plates at 45°C. Revertants appeared at a frequency of 5 x 10-9. Ten revertant strains were taken from separate plates. Contrary to the unstable revertants observed by Morgan et al. which were selected at 420C (19), these revertant strains were stable and did not revert with an appreciable frequency. To determine whether these revertant strains still contained the parental thermolabile glutaminyl-tRNA synthetase, S-100 cell extracts were made and heat-inactivation studies were performed. Incubation at 420C for 5 min reduced the enzyme's activity in the cell extract of all ten revertants to basal level, similar to that observed for the temperature-sensitive parent strain AB4143. This suggested that these temperatureresistant derivatives of AB4143 are revertants by a second site mutation and do not result from back reversion in the ginS gene. Glutaminyl-tRNA synthetase levels in revertant strains. To see if any of the 10 temperature-resistant revertants acquired this phenotype by maintaining a higher level of the thermolabile enzyme, the glutaminyl-tRNA synthetase content in S-100 extracts was determined by antiserum titration. The amount of antiserum needed to remove half of the total enzyme activity in the cell extract of the individual revertant strains was compared to that required for their parent strain AB4143. Only one revertant strain, KL476, showed a fivefold in-

crease in the amount of the enzyme activity (Fig. 1). All other revertants had antiserum titration profiles as for strains AB4143 and AB3441. The results from only one of these revertants, KL477, are illustrated in Fig. 1 to represent them all. The measured specific activity of glutaminyltRNA synthetase in strain KL476 was also about five times higher than that for AB4143 (Table 2). Thus it appears that KL476 may have acquired its temperature resistance by elevating the level of the thermolabile glutaminyl-tRNA synthetase, a mechanism which had been reported earlier for some other aminoacyl-tRNA synthetases (3, 14, 24). Levels of glutamine tRNA in the revertant strains. The level of tRNAGln in the temperature-resistant revertants was detennined by aminoacylating the total tRNA obtained from these strains. One revertant strain, KL477, contained twice as much glutamine-tRNA as did the parent strain AB4143 and the other revertants (Table 2). The level of tRNAGIn in strain KL476 was also shown in Table 2 to represent all other nine revertants. Thus it appears that strain KL477 is temperature resistant by maintaining an elevated level of tRNAGl . This phenomenon was also observed for the ginT mutation (19). Further characterization of these temperature-resistant revertants has concentrated on strains KL476 and KL477. The mutation responsible for the temperature-resistant phenotype of KL476 is designated glnR5 and that for KL477 is designated ginU6.

100 90

80 O-

, 70

a -

60

10

: 50 40

30

20 0

10

20

30

pLI

40 50 60 70 anti-GInRS serum

80

90

100

FIG. 1. Inhibition of glutaminyl-tRNA synthetase activity in ceU extracts of various strains by antiserum. The broken line indicates half inactivation.

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GLUTAMINYL-tRNA SYNTHETASE REGULATORY MUTANTS

TABLE 2. Glutamine tRNA levels and glutaminyltRNA synthetase activity in various strains Relevantgeno type

Gln- tRNA

GlutaminyltRNA thetasesynactivityb

glnSt 30 0.59 ginS 31 0.03 gInS glnR 35 0.16 KL477 ginS glnU 58 0.03 a Picomoles of glutamine accepted per Ame0 unit of

AB3441 AB4143 KL476

tRNA. b Expressed in nanomoles of Gln-tRNA formed per minute at 370C under standard assay conditions by 1 mg of S-100 protein.

Levels of other aminoacyl-tRNA synthetases in strain KIA76. To determine whether the glnR5 mutation led to an increased level of any other aminoacyl-tRNA synthetase in addition to glutaminyl-tRNA synthetase, we measured the specific activity of all other 19 aminoacyl-tRNA synthetases in the aminoacylation reaction. No significant differences in the enzyme levels in strains KL476 and its parent were observed (Table 3). It appears that the one enzyme is selectively overproduced in the revertant strain. Levels of other tRNA's in strain KIA77. Is there a coordinate control of the levels of all or of some tRNA's in strain KL477? To answer this question we determined the levels of the acceptor RNAs for all other 19 amino acids. In addition to tRNAGI, the levels of tRNAMt, tRNA11e, tRNAP", and tRNATrP were also about twice as high as in strain AB4143, and tRNAC38 and tRNAGlU were also elevated significantly in strain KIA77 compared to its parent strain AB4143 (Table 4). Thus, the glnU6 mutation appears to affect the levels of several tRNA

species. Are both tRNAGin species overproduced in strain KL477? In E. coli there are two tRNAGln species. They have base differences in seven positions and can be separated by reversed-phase chromatography (RPC-5). It has been shown by Morgan et al. (19) that the mutationglnToverproduced only tRNAIGl. Therefore we are interested to know which isoacceptor is affected by gin U6. Unfractionated tRNA from strains AB4143 and KL477 was charged with [3H]glutamine and [14C]glutamine, respectively. After cochromatography in RPC-5 the results were normalized to constant tRNA load and plotted (Fig. 2A). Both isoacceptors, tRNA1Gin and tRNA2 in, were elevated to twice the wildtype level. Among the other acceptor RNAs which were elevated in strain KL477, only the isoacceptor

179

pattem of tRNAMet was examined by RPC-5 chromatography (Fig. 2B). Again, it was found that both tRNAMet species of strain KL477 were elevated to twice the wild-type levels. Genetic characterization of strain KIA76 (glnR5). Initial Hfr crosses showed the glnR locus to be between min 23 and 32 on the E. coli TABLE 3. Activity of aminoacylUtRNA synthetases in various strains' Amino acid

AB4143 (gbkS)

KL476(gnR S

Ala 0.2 0.2 Arg 1.5 1.5 Asn 1.7 1.8 Asp 1.4 1.6 0.08 Cys 0.09 Gln 0.03 0.16 Glu 1.4 1.3 Gly 2.8 3.3 His 0.5 0.6 Ile 1.0 1.2 Leu 2.6 2.7 Lys 1.6 1.4 Met 1.9 1.8 Phe 0.6 0.7 Pro 0.3 0.3 Ser 2.9 2.5 Thr 0.2 0.2 Trp 0.12 0.16 Tyr 0.4 0.4 Val 1.5 1.5 'Data expressed in nanomoles of aminoacyl-tRNA formed per minute at 370C under standard assay conditions by 1 mg of S-100 protein. TABLE 4. Level of amino acid acceptor RNAs in the unfractionated tRNA in various strainsa Amino acid

AB4143 (ginS)

KL477 (ghS

Ala

87 102 80 85 39 30 100 129 72 36 99 75 45 62 18 78 98 29 55 82

87 98 78 90 68

Arg Asn Asp Cys Gln Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr

Trp Tyr

U8 133 129 72 72 105 71 90 60 35 72 100

59 49 Val 90 aData expressed as picomoles per Am60 unit.

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6

2

5

10

O

4

a8

C

3

a

14

0-

z

0-

c 6 0

4

2.

z

2 90

94

98 102 106 Fraction number

110

I 60

68

64

72

76

80

84

88

92

96

Fraction number

FIG. 2. Reversed-phase chromatography (RPC-5) of unfractionated [3H]aminoacyl-tRNA from strain AB4143 and [14Claminoacyl-tRNA from strain KL477. (A) Gln-tRNA; (B) Met-tRNA. An equal amount of 3H and '4C radioactivity (representing different amounts of unfractionated tRNA in each sample) was applied to the column. The eluted radioactivity was then normalized to equal tRNA load (1 Ameb unit) and converted into picomoles of tRNA.

chromosome. In using F15/KL476 to introduce glnR5 into strain KL304 (ginS purB), a high linkage of glnR5 and purB+ (ca. 95%) was observed. P1 grown on KL476 (glnS glnR5 pyrC+

purB')

was

therefore used to transduce strain

TABLE 5. Pl transduction linkage ofglnR with pyrC and purB

DnrRecipient relevlt

(reevant geno-

(rlvn

Total

Se trans- Classes of transducle etd duetants (%

markertas type) KL481 (glnS pyrC purB), and the transduction (0) data are presented in Table 5. When purB+ KL476 KL481 purB+ 173 glnR+ pyrC+ (glnS (ginS transductants were selected, the frequency of glnR glnRcotransduction of ginR was 83%. The cotranspurB+ purB pyrC+) pyrC) duction frequency of ginR with pyrC+ was 42%. glnR+ pyrC (30) A gene order ofpyrC-ginR-purB could be estabglnRpyrC+ (0) lished, which places glnR at approximately mim glnR pyrC (143) 24 on the E. coli map (2) (Fig. 3). pyrC+ 118 glnR+ purB+ (0) Genetic characterization of strain KIA77 glnR+ purB (69) glnR purB+ (0) (glnU). Initial crosses of strain KL477 with varglnR purB (49) ious Hfr strains (16) showed unambiguously that ginU maps counterclockwise and very close to TABLE 6. Pl transductional linkage ofglnU to the origin of strain KL16. To get finer map cysC and argA position of ginU, KL479 was crossed with Hfr KL267, which transfers thyA+, argA, and cysC Donor Recipient SeTotal early. This cross indicated that glnU is linked to (relevant (relevant lected trans- Classes of tranaducductants cysC and to argA and also gave rise to a recom- genotype) genotype) marker tants binant (KL480) which maintains the tempera- W3110 KL480 cysC+ 270 glnU+ (26) ture-resistant character of KL477 but also car(glnS+ (ginS argA+ ries cysC and argA alleles. P1 grown on W3110 glnU+ glnU cysC+ cM8C was used to transduce KL480. Both cys+ and argA +) argA) arg+ transductants were analyzed (Table 6). ginU+ argA (101) From the data, it can be seen that the wild-type glnU argA+ (7) allele of glnU has a cotransduction frequency of ginU argA (122) argA+ 138 glnU+ cysC+ (31) 73% with argA and 50% with cysC. From the glnU+ cysC (70) relative frequencies of single- and double-crossglnUcysC+ (16) over classes (Table 6), the map order of argAglnU cysC (21) gln U-cysC could be deduced. This located glnU at about min 59.5 on the E. coli map (2) (Fig. 3). suppress the temperature sensitivity of strains Physiological characterization of KIA76 KL476 and 477 caused by the thermolabile gluand KIA77. The ginR and glnU mutations can taminyl-tRNA synthetase. It was therefore ingenotype)

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181

100 0

xyl

Str A

FIG. 3. The circular linkage map of E. coli with the markers of interest for this study. The scale and genetic symbols are according to those of Bachmann et al. (2). A, The point of insertion and direction of transfer of Hfr strains which were used in the initial genetic characterization of strains KL476 and KL477.

teresting to determine if these mutations affected the levels of aminoacyl-tRNA, the synthesis of the cognate amino acid, and the growth characteristics of the strains. (i) Levels of glutaminyl-tRNA in vivo. To correlate the growth rates with the levels of aminoacyl-tRNA, the in vivo level of gln-tRNA in strains AB3441, AB4143, KL476, and KL477 was determined by using acetylated DBAE-cellulose chromatography (17). It was found that at 390C (near the nonpermissive temperature for strain AB4143 in minimal medium), only 28% of the total tRNAGIn in strain AB4143 was aminoacylated, revealing the effect of the thermolabile glutaminyl-tRNA synthetase. In the temperature-resistant strains KL476 and KL477, the percentage of tRNAGlN) aminoacylated and present in vivo was 75 to 80%, similar to the level found in the wild type-strain AB3441 (Table 7). It is interesting to see that even in strain KL477, which has twice the amount of tRNAG'n as wildtype strains, also the same high percentage (74%) of its tRNAGI, was aminoacylated in vivo. Similar measurements were made for these strains grown at 300C, and the level of Gln-tRNA remained similar in all cases. Thus, the glnR and gln U mutations overcome the temperature sen-

sitivity by providing enough gln-tRNA to sustain protein synthesis, while with glnS alone, the supply of gln-tRNA is very restricted at 390C. Furthermore, the level of gln-tRNA in the various strains at different temperatures also correlated well with their doubling times at these temperatures (Table 7). As a control the levels of Met-tRNA (80% aminoacylated) were the same in all strains. Although strain KL477 contains twice as much tRNAmet as does wild type, the percentage of charged tRNA was as high as in wild type strains (data not shown). Possibly there is a general optimal charging level of tRNA around 75 to 80%, and this may be related to the accuracy of protein synthesis. (ii) Glutamine biosynthesis and intracellular glutamine concentration. Since the involvement of aminoacyl-tRNA in the regulation of the biosynthesis of their cognate amino acid (4, 13) is well known in some cases, we examined the question of a possible correlation between the level of glutaminyl-tRNA and the biosynthesis of glutamine. Both the transfer reaction (26) and the phosphate release assay (26) were used to determine the total amount and the biosynthetic activity, respectively, of glutamine synthetase. The amount of glutamine synthetase

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CHEUNG ET AL.

TABLE 7. Some physiological measurements ofAB3441, AB4143, KL476, and KL477 In vivo

glntRNdA

Doubling time (min)

mg

Strain (relevant genotype) LB

AB3441 (glnS+) AB4143 (gInS) KLA76 (ginS ginR) KL477 (ginS giU) a

b

Glutamine synthetase (units/

ophosphate

Minimal

370C 45°C 300C 390C 30 36 66 50 60 _c 60 100 50 45 69 65 55 48 72 75

30°C 39°C Amta

Actb

0.40 0.28 0.80 0.31

0.26 0.19 0.16 0.19

65 50 65 58

71 28 80 74

Histidinol

phosphatase (units/10' cells) 0.08 0.10 0.08 0.07

(Gmol/109 cne(ml/lO' cells)

conc

4 47 6 5

Transferase assay.

Phosphate release assay. -, No growth.

was elevated in strain KIA76 but not strain KLA77 at 39°C, where the level of Gln-tRNA was high in both strains (Table 7). The biosynthetic activity of glutamine synthetase remained the same in all strains. As an unrelated enzyme,

the activity of histidinol phosphate phosphatase was also assayed and found to be the same for these strains. Thus there was no general repression-derepression phenomenon. When the concentration of free amino acid was examined in these four strains, strain AB4143 had a 20-fold higher glutamine level than found in any of the others. It is difficult to attribute the high glutamine concentration in strain AB4143 to the inability of the thermolabile glutaminyl-tRNA synthetase to utilize it. A better correlation between the levels of Gln-tRNA and glutamine metabolism may be made when other enzymes and intermediates involved, such as glutamate synthase, glutamate dehydrogenase, and a-ketoglutarate, have been measured (25). DISCUSSION selection scheme described by Morthe Using gan et al. (19), we have isolated two mutations in E. coli, one of which affects the level of glutaminyl-tRNA synthetase (gln)), while the other controls the level of tRNAGlIn and several other tRNA species (ginU). The ginR mutation increases only the level of glutaminyl-tRNA synthetase; other aminoacyltRNA synthetases are not affected by it. The total amount of tRNAGInI, as well as the relative amounts of the two isoacceptors, are not altered in a ginR strain. Thus tRNAGln and glutaminyltRNA synthetase are not regulated together. Similar results have been observed in other regulatory mutations affecting leucyl- (12, 24), seryl- (24), and valyl-tRNA synthetases (3). Our search for the possibility of a coordinate control of all or a few aminoacyl-tRNA synthetases has not yet shown the existence of such a control,

although a common regulation of the levels of the aminoacyl-tRNA synthetases for the three branched-chain amino acids has been reported (10). Since ginS, the structural gene for glutaminyltRNA synthetase, and ginR are at well-separated genetic locations, the ginR gene product must be trans-acting and is probably a diffusible compound. The glnR5 mutation is not affected by the presence of supE in the strain nor suppressed by the amber suppressor, supD, or the ochre suppressor, supG, when they were genetically introduced into strain KL476 (data not shown). Nevertheless, the failure of the above suppressors to suppress glnR5 does not rule out the possibility that this gene codes for a protein as has been shown for the leuY, leuR, and serR mutations (11, 24). The ginR5 mutation appears to affect only the thermolabile glutaminyl-tRNA synthetase. When a wild type ginS allele was introduced into strain KL476, the level of this enzyme remained the same as in the wild-type strain AB3441 (data not shown). It appears that the decision to maintain a particular level of functional glutaminyl-tRNA synthetase may depend on the cell's ability to keep the normally high ratio of charged to uncharged tRNAGlI; ginR does not constitutively cause the glutaminyltRNA synthetase to stay at a higher than normal level. We have been cautious in interpreting our observations of an increased level of glutaminyltRNA synthetase. The observed level could be a result of overproduction at the transcriptional or translational level. Altematively, it could also be a result of a decreased degradation rate. Baer et al. (3) have shown that in the case of valR, the increased level of valyl-tRNA synthetase observed is a result of overproduction and not a decreased degradation rate. For the other similar mutations studied, e.g., leuY, leuR, and serR,

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GLUTAMINYL-tRNA SYNTHETASE REGULATORY MUTANTS

the above two possibilities have not been distinguished (12, 24). One interesting result observed for glnR is its pleiotropic effect on glutamine synthetase production. ginR not only resulted in an elevated level of glutaminyl-tRNA synthetase, but also that of glutamine synthetase, the enzyme that makes glutamine. Although there is evidence in some cases for a participation of aminoacyltRNA's in the regulation of the biosynthesis of their cognate amino acids (4, 13), there does not seem to be a direct correlation between the level of Gln-tRNA, glutamine synthetase, and free glutamine when the results of the wild-type strain AB3441, the temperature-sensitive parent AB4143, and the two temperature-resistant revertants KL476 and KL477 were compared (Table 7). However, glutamine metabolism is complex, and glutamine synthetase is a complicated enzyme all by itself. To understand the observed changes in glutamine synthetase in KL476, other parameters, such as the levels of glutamate synthase, a-ketoglutarate, and glutamate dehydrogenase have to be measured (25). The gln U6 mutation causes a twofold increase in tRNAGI. This higher tRNA concentration possibly offers substrate protection to the thermolabile cognate glutaminyl-tRNA synthetase to render the cells temperature resistant as discussed for the ginT mutation (19). Contrary to the ginT mutation which affects only tRNAGln ginU increases the level of tRNAG'n, tRNAMet tRNAne, tRNATm, and tRNAPro to similar extents (i.e., twofold) and raises that of tRNAGlU and tRNACYS somewhat above the normal level. It is difficult to interpret the sipificance of the data on the level of these tRNA species. If the increased overall level were due to an unproportionately high percentage of one of the isoacceptors, this result would be overlooked. RPC-5 chromatography is very useful in resolving isoacceptors of tRNA's (21). Contrary to the ginT mutation in which only the level of one of the two glutaminyl-tRNA isoacceptors is elevated, ginU causes both of them to be elevated to similar extent (Fig. 2A). Furthermore, all the isoacceptors of tRNAMet are also elevated to similar extent (Fig. 2B). Possibly all isoacceptors are proportionately increased for those acceptor RNAs that are present in higher levels in strain KL477. However, there is no detectable overproduction of total tRNA in this strain because the amount of tRNA isolated from a certain amount of cells was the same in the mutant and the parent strains. The nature of glnU is interesting. Ikemura et al. (9) reported that tRNA/M't maps in the vicinity of this mutation (around min 60) on the E.

183

coli genetic map. None of the other tRNA species affected by ginU in strain KL477 was localized by these authors in this region. Yet, they may still be present there since the complete arrangement of tRNA genes on the E. coli chromosome is not known. In this case the ginU mutation could be of an operator-promoter nature controlling the level of expression of these tRNA's on a multimeric gene cluster. Indeed, when an episome carrying glnUW (F143/1) was introduced into strain KIA77, the temperatureresistant phenotype caused by glnU was dominant. Thus glnU may be cis-acting. Furthermore, ghnU cannot discriminate between the presence of a wild-type or a thermolabile glutaminyl-tRNA synthetase. When glnS' was introduced into strain KL477, the tRNA species which were affected byglnUin KIA77 remained at high levels in the presence of a wild-type glutaminyl-tRNA synthetase (data not shown). However, the data do not rule out the possibility that ginU is a mutation in a regulatory protein. The fact that most tRNAGlZn genes are located elsewhere (9, 19) would require a very high expression of the hypothetical tRNAGlI genes near ginU. In addition, the fact that all tRNAMt isoacceptors are overproduced and that a variety of other tRNA's are affected (to a lesser extent) may indicate that ginU specifies a regulatory protein. This intriguing protein would cause selective tRNA gene transciption or selective processing/degradation of tRNA precursors. Although gin U maps near relA and reiX, two genes involved in stable RNA regulation, this mutation does not change the Rel character of the strains (data not shown). As in ginT mutation (19) the elevated tRNA levels in the gin U strain do not cause a concomitant increase in the levels of the cognate aminoacyl-tRNA synthetase. This finding complements the results of the effects of synthetase overproduction discussed above. The ginR and ginU mutations described in this paper are further examples of the existence of regulatory mechanisms for aminoacyl-tRNA synthetases and tRNA's. To unravel the details of these processes, much greater knowledge of the number, organization, and structure of all tRNA genes in E. coli and of the involvement of aminoacyl-tRNA in regulatory processes needs to be assembled. ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health and the National Science Foundation.

LITERATURE CITED 1. Bachmann, B. J. 1972. Pedigrees of some mutant strains

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of Escherichia coli K-12. Bacteriol. Rev. 36:525-557. 2. Bachmann, B. J., K. B. Low, and A. L, Taylor. 1976. Recalibrated linkage map of Escherichia coli K-12. Bacteriol. Rev. 40:116-167. 3. Baer, ML, K. B. Low, and D. S611. 1979. Regulation of the biosynthesis of aminoacyl-transfer ribonucleic acid synthetases and of transfer ribonucleic acid in Escherichia coli. V. Mutants with increased levels of Valyltransfer ribonucleic acid synthetase. J. Bacteriol. 139: 165-175. 4. Brenchley, J. E., and L. S. Williams. 1975. Transfer RNA involvement in the regulation of enzyme synthesis. Annu. Rev. Microbiol. 29:251-274. 5. Britten, R. J., and F. T. McClure. 1962. The amino acid pool in Escherichia coli. Bacteriol. Rev. 26:292-335. 6. Clarke, S. J., B. Low, and W. H. Konigsberg. 1973. Close linkage of the genes serC (for phosphohydroxy pyruvate transamine) and serS (for seryl-transfer ribonucleic acid synthetase) in Escherichia coli K-12. J. Bacteriol. 113:1091-1095. 7. Ely, B. 1974. Physiological studies of salmonella histidine operator-promoter mutants. Genetics 78:593-606. 8. Hayashi, S., J. P. Koch, and E. C. C. Lin. 1974. Active transport of L-a-glycerophosphate in Escherichia coli. J. Biol. Chem. 239:3098-3105. 9. Ikemura, T., and H. Ozeki. 1977. Gross map location of Escherichia coli transfer RNA genes. J. Mol. Biol. 117: 419-446. 10. Jackson, J., L, S. Williams, and H. E. Umbarger. 1974. Regulation of synthesis of the branched-chain amino acids and cognate aminoacyl-transfer ribonucleic acid synthetases of Escherichia coli: a common regulatory element. J. Bacteriol. 120:1380-1386. 11. Korner, A., B. B. Magee, B. Liska, K. B. Low, E. A. Adelberg, and D. S611. 1974. Isolation and partial characterization of a temperature-sensitive Escherichia coli mutant with altered glutaminyl-transfer ribonucleic acid synthetase. J. Bacteriol. 120:154-158. 12. LaRossa, R. A., J. Mao, K. B. Low, and D. S11. 1977. Regulation of biosynthesis of aminoacyl-tRNA synthetases and of tRNA in Escherichia coli. III. Biochemical characterization of regulatory mutants affecting leucyltRNA synthetase levels. J. Mol. Biol. 117:1049-1059. 13. LaRossa, R., and D. S611. 1978. Other roles of tRNA, p. 136-167. In S. Altman (ed.), Transfer RNA. MIT Press,

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14. LaRossa, R., G. Vogeli, K. B. Low, and D. 8611. 1977. Regulation of biosynthesis of aminoacyl-tRNA synthetases and of tRNA in Escherichia coli. II. Isolation of regulatory mutants affecting leucyl-tRNA synthetase levels. J. Mol. Biol. 117:1033-1048. 15. Lloyd, R. G., B. Low, G. N. Godson, and E. H. Birge.

J. BACTERIOL. 1974. Isolation and characterization of an Escherichia coli K-12 mutant with a temperature-sensitive RecAphenotype. J. Bacteriol. 120:407415. 16. Low, B. 1973. Rapid mapping of conditional and auxotrophic mutations in Escherichia coli K-12. J. Bacteriol. 113:798-812. 17. McCutchan, T. F., P. T. Gilham, and D. Sll. 1975. An improved method for the purification of tRNA by chromatography on dihydroxyboryl substituted cellulose. Nucleic Acid Res. 2:853-864. 18. Morgan, E. A., T. Ilkemura, L. E. Post, and M. Nomura. 1979. tRNA genes in ribosomal RNA operons of Escherichia coli. In J. Abelson, P. Schimmel and D. Soll (ed.), Transfer RNA. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 19. Morgan, S., A. Korner, K. B. Low, and D. 8611.1977. Regulation of biosynthesis of aminoacyl-tRNA synthetases and of tRNA in Escherichia coli. I. Isolation and characterization of a mutant with elevated levels of tRNA,GI". J. Mol. Biol. 117:1013-1031. 20. Morgan, S., and D. Sll. 1978. Regulation of the biosynthesis of amino acid:tRNA ligases and of tRNA. Prog. Nucleic Acid Res. Mol. Biol. 21:181-207. 21. Pearson, R. L, J. F. Weiss, and A. D. Kelmers. 1971. Improved separation of transfer RNAs on polychlorotrifluoroethylene-supported reversed-phase chromatography columns. Biochim. Biophys. Acta 228:770-774. 22. Schimmel, P. R., and D. So1. 1979. Aminoacyl-tRNA synthetases. General features and recognition of transfer RNAs. Annu. Rev. Biochem. 48:601-648. 23. S611, D., and Schimmel, P. R. 1974. Aminoacyl-tRNA synthetases, p. 489-538. In P. Boyer (ed.), The enzymes, vol. X. Academic Press Inc., New York. 23a.Steege, D., and D. S611. 1979. Suppression, p. 433-485. In R. Goldberger (ed.), Biological regulation and development. I. Gene expression. Plenum Publishing Co., New York. 24. Theall, G., K. B. Low, and D. 861. 1979. Regulation of the biosynthesis of aminoacyl-tRNA synthetases and of tRNA in Escherichia coli. IV. Mutants with increased levels of leucyl- or seryl-tRNA synthetase. Mol. Gen. Genet. 169:205-211. 25. Tyler, B. 1978. Regulation of the asimilation of nitrogen compounds. Ann. Rev. Biochem. 47:1127-1162. 26. Woolfolk, C. A., B. M. Shapiro, and E. R. Stadtman. 1966. Regulation of glutamine synthetase. I. Purification and properties of glutamine synthetase from Escherichia coli. Arch. Biochem. Biophys. 116:177-192. 27. Yaniv, M., and W. R. Folk. 1975. The nucleotide sequences of the two glutamine transfer ribonucleic acids from Escherichia coli. J. Biol. Chem. 250:3243-3253.

Regulation of the biosynthesis of aminoacyl-transfer ribonucleic acid synthetases and of transfer ribonucleic acid in Escherichia coli. VI. Mutants with increased levels of glutaminyl-transfer ribonucleic acid synthetase and of glutamine transfer ribonucleic acid.

Vol. 139, No. 1 JOURNAL OF BACRziOLOGY, July 1979, p. 176-184 0021-9193/79/07-0176/09$02.00/0 Regulation of the Biosynthesis of Aminoacyl-Transfer R...
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