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J Bacteriol. Apr 1998; 180(7): 1808–1813.
PMCID: PMC107094

The Methyl Group of the N6-Methyl-N6-Threonylcarbamoyladenosine in tRNA of Escherichia coli Modestly Improves the Efficiency of the tRNA

Abstract

tRNA species that read codons starting with adenosine (A) contain N6-threonylcarbamoyladenosine (t6A) derivatives adjacent to and 3′ of the anticodons from all organisms. In Escherichia coli there are 12 such tRNA species of which two (tRNAGGUThr1 and tRNAGGUThr3) have the t6A derivative N6-methyl-N6-threonylcarbamoyladenosine (m6t6A37). We have isolated a mutant of E. coli that lacks the m6t6A37 in these two tRNAGGUThr species. These tRNA species in the mutant are likely to have t6A37 instead of m6t6A37. We show that the methyl group of m6t6A37 originates from S-adenosyl-l-methionine and that the gene (tsaA) which most likely encodes tRNA(m6t6A37)methyltransferase is located at min 4.6 on the E. coli chromosomal map. The growth rate of the cell, the polypeptide chain elongation rate, and the selection of Thr-tRNAGGUThr to the ribosomal A site programmed with either of the cognate codons ACC and ACU were the same for the tsaA1 mutant as for the congenic wild-type strain. The expression of the threonine operon is regulated by an attenuator which contains in its leader mRNA seven ACC codons that are read by these two m6t6A37-containing tRNAGGUThr species. We show that the tsaA1 mutation resulted in a twofold derepression of this operon, suggesting that the lack of the methyl group of m6t6A37 in tRNAGGUThr slightly reduces the efficiency of this tRNA to read cognate codon ACC.

All tRNA species from the three domains, Archaea, Bacteria, and Eucarya, contain modified nucleosides, which are derivatives of the four nucleosides, adenosine, guanosine, cytidine, and uridine. At present, more than 79 different modified nucleosides from the tRNA of various organisms have been characterized (23). Some of these are present in tRNA from only one domain, but a few are present in the same subset of and at the same position in the tRNAs from all three domains (3). One such conserved group of modified nucleosides is the threonylated adenosine (t6A) derivatives. These modified adenosines are present adjacent to and 3′ of the anticodon (position 37) in the subset of tRNAs that reads codons starting with A. The universal presence of t6A derivatives suggests that these kinds of modifications may have been present in the tRNA of the progenitor, unless a convergent evolution has occurred. This conservation also suggests that the functions of these modified nucleosides may be principally the same in all organisms.

In Escherichia coli, the t6A37 derivative N6-methyl-N6- threonylcarbamoyladenosine (m6t6A37) is present in only two tRNA species, the tRNAGGUThr species, with the same anticodon (20). Threonine is the precursor in the synthesis of t6A (10, 32), and in vitro threonylation requires carbonate and ATP (15, 21). Here we show that the methyl group of m6t6A37 originates from methionine. So far, no mutant deficient in any t6A37 derivative has been characterized. As a first step to elucidate the syntheses of these groups of modified nucleosides and their roles in vivo, we have isolated and characterized a mutant deficient in the synthesis of m6t6A37. We show that the tsaA gene most likely encodes the tRNA(m6t6A37)methyltransferase that transfers a methyl group from S-adenosylmethionine (AdoMet) to the two tRNAGGUThr species containing the t6A moiety. The tsaA gene was localized to the 4.6 min site on the E. coli chromosome. We also show that the methyl group of m6t6A37 slightly improves the translational efficiency of the two tRNAGGUThr species.

MATERIALS AND METHODS

Bacteria, growth conditions, and genetic procedure.

Bacterial strains used were all derivatives of E. coli K-12 and are listed in Table Table1.1. Cultures were grown in either Luria-Bertani (LB) medium (1) or nutrient broth (0.8%; Difco Laboratories, Detroit, Mich.) supplemented with 0.5% NaCl. The minimal medium was made from the basal medium (medium E) described by Vogel and Bonner (40) supplemented with 0.2% glucose, thiamine (1 μg/ml), and required amino acids (50 μg of the l isomer per ml). In some experiments the MOPS (morpholinepropanesulfonic acid)-glucose minimal medium (28) and M9 medium (25) were used. Kanamycin and carbenicillin were used at 50 μg/ml. The Hfr mapping procedure was adapted from Singer et al. (39). P1 transduction was done as described by Miller (25).

TABLE 1
Bacterial strains and plasmids

Analysis of modified nucleosides in tRNA.

Cells were grown in 45 ml of LB medium, harvested at a cell density of about 4 × 108 cells/ml by centrifugation, and washed with 0.9% NaCl. tRNA was prepared by lithium chloride fractionation (9) and degraded to nucleosides by nuclease P1 and alkaline phosphatase (17). The hydrolysate was then analyzed by high-performance liquid chromatography (HPLC) according to the method of Gehrke and Kuo (17). For the analysis of methylated nucleosides by thin-layer chromatography, cells were grown in rich MOPS medium (27) in the presence of l-[methyl-14C]methionine (0.074 μg/ml; 20 or 55 μCi/μM). The methyl-14C-labelled tRNA was degraded to nucleosides as described previously (2) and analyzed by thin-layer chromatography as described by Rogg et al. (34). The various radioactive compounds were located by autoradiography, and the radioactivity in each compound was determined by scintillation counting (2).

Determination of tRNA methyltransferase activity in vitro.

Cells were grown in 50 ml of LB medium at 37°C and harvested at a density of 3 × 108 cells/ml. The cells were pelleted, washed twice with 0.9% NaCl and once with buffer A (25 mM Tris-HCl [pH 7.4], 10 mM magnesium acetate, 0.1 mM dithiothreitol, 1 mM EDTA, 10% [vol/vol] ethylene glycol), and resuspended in 0.5 ml of buffer A. Cells were disrupted by sonication three times for 5 s at 20% power on a VCX400 sonicator (Sonics & Materials Inc., Danbury, Conn.). Cell debris was removed by centrifugation at 15,000 rpm with a Beckman JA-20 rotor for 10 min at 4°C, and the supernatant was transferred to a new tube. Ribosomes were removed by ultracentrifugation at 300,000 × g. The obtained supernatant was used as an enzyme source. The reaction mixture contained 100 μg of bulk tRNA from strain GRB1109 (tsaA1) or strain GRB1108 (tsaA+), 80 μl of enzyme extract, 10 μl of [methyl-14C]AdoMet (60 μCi/μM), 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM NH4Cl, and 0.1 mM dithiothreitol in a final volume of 1 ml. The mixture was incubated for 3 h at 37°C. The reaction was stopped by adding 1 ml of phenol saturated with water followed by vigorous shaking for 10 min at room temperature. RNA in the aqueous phase was precipitated by ethanol. The precipitate was washed twice with 80% ethanol, dried, and digested either to nucleotides or to nucleosides. The distribution of methylated nucleotides or nucleosides was determined by two-dimensional thin-layer chromatography as described by Nishimura (30) and Rogg et al. (34), respectively. The radioactive compounds were detected by a PhosphorImager (Molecular Dynamics) (data not shown). Meanwhile, the rest of the nucleoside sample was assayed by HPLC and the radioactivity in the eluate was monitored by a flow scintillation analyzer (Radiomatic FLO-ONE beta; Packard Instrument Co., Meriden, Conn.) (Fig. (Fig.22C).

FIG. 2
HPLC of nucleosides from tRNA from strain GRB1108 (tsaA+) (A) and strain GRB1109 (tsaA1) deficient in m6t6A (B). tRNA was degraded to nucleosides and a part (retention time, 0 to 80 min) of the chromatogram is shown. No difference in the patterns ...

Determination of sensitivity to various amino acid analogs.

Strains GRB1108 (tsaA+) and GRB1109 (tsaA1) were tested for sensitivity to amino acid analogs as described by Ericson and Björk (16) and Cortese et al. (11). All the 27 analogs specified in reference 16 were obtained from Sigma Chemical Co., St. Louis, Mo.

Determination of growth and polypeptide chain elongation rates.

Growth rates at 37°C in rich MOPS medium and three MOPS minimal media with three different carbon sources (glucose, glycerol, and acetate) were determined as described by Björk and Neidhardt (5). Polypeptide chain elongation rates were determined as described by Ericson and Björk (16) and Schleif et al. (36).

Determination of ribosomal A-site selection and P-site frameshifting.

The rate of aminoacyl-tRNA selection to the A site and the P-site frameshifting ability were determined as described earlier (13, 14, 18).

RESULTS

Strain GRB105 lacks three modified nucleosides in its tRNA.

During the analysis of various aro strains of E. coli for the presence of two modified nucleosides (cmo5U and its methylester, mcmo5U) (2), we noticed that one such strain (RE113 [aroE]), in addition to cmo5U and mcmo5U deficiencies (due to the aroE mutation) also lacks an unknown methylated nucleoside denoted no. 5 in Fig. 1 of reference 2. A spontanous Strr derivative of strain RE113 was isolated for future Hfr mapping. This strain (GRB105 [aroE strL]) was grown in rich MOPS medium containing l-[methyl-14C]methionine. tRNA was prepared, digested to nucleosides, and analyzed by two-dimensional thin-layer chromatography (34). Like strain RE113, strain GRB105 also lacked compound no. 5 (2). Further analyses suggested that compound no. 5 may be m6t6A. First, m6t6A migrates similarly to our compound no. 5 (34). Furthermore, compound no. 5 becomes radioactively labelled when wild-type cells are grown in the presence of either [14C]HCO3 or [14C]threonine, conditions that are known to label m6t6A (15).

For the current work, tRNA from strain GRB105 was digested to nucleosides and analyzed by HPLC. This analysis revealed that this tRNA was lacking, besides the cmo5U and mcmo5U nucleosides, three other modified nucleosides: 2-thiocytidine (s2C), 2-methyladenosine (m2A), and an unknown modified nucleoside. The migration properties and UV spectrum of the unknown modified nucleoside suggested that it was m6t6A (data not shown). Below we strengthen the conclusion that the unknown nucleoside is m6t6A. Thus, strain GRB105 contained at least three mutations, stcA1, trmG1, and tsaA1, that affect the syntheses of s2C, m2A, and probably m6t6A, respectively.

The tsaA gene is located at 4.6 min on the E. coli chromosome.

We used strain GRB105 (aroE stcA1 trmG1 tsaA1 strL) in Hfr crosses with strains (39) that have Tn10 at defined locations to localize the tsaA1 mutation to the region between 96 and 7 min (between cysA [96.5 min] and zag-3198::Tn10 [6.8 min]). We scored the allelic states of genes tsaA, stcA, and trmG from recombinants by HPLC analysis of the modification patterns of tRNA. The stcA and trmG genes are located in the regions from 28 to 36 min and from 10 to 28 min, respectively, (33a) and were not further characterized.

To further refine the map position of the tsaA gene, we used various Tn10 or Tn10kan insertions located in the 0- to 6.5-min region to determine whether any of them were tightly linked to the tsaA gene. We found that the tsaA mutation was 75% linked (nucleoside compositions of tRNA from 20 recombinants were analyzed by HPLC) to the zae-3095::Tn10kan at 4.75 min. We then made a three-factor cross to establish the gene order in this region. We used strain GRB928 (proAB8::Tn10) as the donor and strain GRB1037 (tsaA1 zae-3095::Tn10kan) as the recipient. The phenotypes of 100 Tetr transductants were monitored on plates (Km phenotype) and by HPLC of degraded tRNA. Our results suggest that the gene order is tsaA, zae, proAB (Fig. (Fig.1).1). When the tsaA phenotype is ignored, zae-3095::Tn10kan and proAB81::Tn10 are only weakly linked (7% cotransduction). However, the zae-3095::Tn10kan allele always cotransfers in tsaA1 proAB::Tn10 cotransductants. These results suggest that zae-3095::Tn10kan is located between tsaA and proAB. Together, these data place the tsaA gene at 4.6 min on the E. coli map (Fig. (Fig.1).1). By using one of the transductants (GRB1037 [aroE strL zae-3095::Tn10kan tsaA1]) as the donor and strain CAG18447 as the recipient, the congenic strains GRB1108 (tsaA+) and GRB1109 (tsaA1) were constructed.

FIG. 1
Mapping of the tsaA gene by P1 transduction. A physical map of the donor and recipient is shown. Strain CAG18447 (proAB81::Tn10) was used as the donor, and ...

The tsaA gene probably encodes tRNA(m6t6A37)methyltransferase.

To further characterize the tsaA1-related nucleoside deficiency, tRNA was prepared from the two congenic strains GRB1108 (tsaA+) and GRB1109 (tsaA1) and digested to nucleosides. Figure Figure2A2A and B show that the only difference in the tRNA modification patterns of the wild-type tsaA+ and the mutant tsaA1 observed was due to the compound that we now identify as m6t6A. We identify this compound as m6t6A by the following criteria. (i) Its migration in the chromatography system devised by Gehrke and Kuo (17) is the same as that observed for m6t6A in their analysis. (ii) The UV spectrum is the same as that published for m6t6A. (iii) By mass spectroscopy, the molecular weight of the unknown compound was found to be 427 as expected for the protonated form of m6t6A. Only two tRNA species, tRNAGGUThr1 and tRNAGGUThr3 in E. coli, have m6t6A37 (20). If only the methyl group were lacking in the mutant tRNAGGUThr, we would expect an increase in the level of t6A in bulk tRNA. Indeed, the level of t6A in the mutant increased (the ratio of t6A to m2A, measured as the absorbancy at 254 nm, increased from 0.71 in the wild type to 0.96 in the tsaA1 mutant). Together, these data suggest that tsaA1 prevents the methylation of t6A.

If the tsaA1 mutation were responsible for the addition of the methyl group of m6t6A in tRNAGGUThr, then one would expect that the tRNA would serve as a substrate for in vitro methylation. We therefore tested whether the tRNA from strain GRB105, which lacks both m2A and m6t6A, was able to accept methyl groups in vitro. tRNA from the tsaA1 mutant was mixed with enzyme extract from the wild-type strain carrying tsaA+; [methyl-14C]AdoMet was the methyl donor. Following incubation at 37°C, the tRNA was reisolated and degraded to nucleosides or nucleotides and the mixture was analyzed by thin-layer chromatography and HPLC, respectively (Fig. (Fig.2C).2C). Unexpectedly, no radioactivity was detected in the m2A area of the two-dimensional thin-layer chromatogram, suggesting that tRNA(m2A37)methyltransferase was not active in vitro (data not shown). However, the compound migrating as m6t6A contained radioactivity, as also shown in HPLC analysis (Fig. (Fig.2C).2C). As expected, no radioactivity in the region to which m6t6A migrates was observed when tRNA from wild-type strain GRB1108 was analyzed in a similar way (data not shown; see the legend for Fig. Fig.2C).2C). Since neither ATP, l-threonine, nor HCO3, which all are required for in vitro threonylation (15), was added to the incubation mixture, it is unlikely that the threonine moiety was added in vitro. Thus, it is likely that the product of the tsaA gene is tRNA(m6t6A37)methyltransferase and that the two tRNAGGUThr species have t6A37 instead of m6t6A37 in the tsaA1 mutant.

Lack of m6t6A37 does not affect the growth rate, the polypeptide step time, or the response to 27 amino acid analogs.

Several tRNA modification-deficient mutants grow slower than the wild type and translate mRNA at a reduced rate (4). Therefore, we investigated whether the lack of m6t6A37 also reduced the growth rate and the polypeptide chain elongation rate. Steady-state cultures of the congenic pairs GRB1108 (tsaA+) and GRB1109 (tsaA1) were prepared in MOPS-glucose, MOPS-glycerol, MOPS-acetate, and rich MOPS media at 37°C. We did not observe any differences between the growth rates of the wild-type control and the tsaA1 mutant in any of these media (data not shown). Furthermore, we also determined the time by which ribosomes translate the lacZ mRNA (16, 36). No difference between the tsaA1 mutant and the wild-type control was observed. It has been shown earlier that a deficiency in Ψ38, Ψ39, Ψ40 (11), ms2io6A37 (16), or m1G37 (22) results in an altered response to various amino acid analogs, suggesting that expression of the corresponding biosynthetic enzymes may be altered (see reference 4 for a recent review). We therefore tested 27 different amino acid analogs (specified in reference 16), but no effect was observed with any of the tested analogs. Note, however, that no threonine analog was tested.

Lack of the methyl group of m6t6A37 does not induce +1 frameshifting at an ACC-A or ACC-U site and does not influence the Thr-tRNA selection to the ribosomal A sites at ACC and ACU codons.

There is precedent for the possibility that a deficiency in a modified nucleoside, such as m1G37 or ms2i6A37, may cause the tRNA to make +1 frameshift errors (6, 18, 33, 38). In Salmonella typhimurium, a +1 frameshift suppressor (sufJ105) was isolated and shown to be a derivative of tRNAGGUThr3 (8). The altered tRNAGGUThr3 promotes +1 frameshifting at sites in the mRNA where any of the four-base sequences ACC-A, ACC-U, and ACC-C is encountered (7). (The ACC codon is in the zero frame, and a +1 frameshift moves the reading frame one step to the right, e.g., to the CC-A triplet.) Since m6t6A37 is present in the wild-type form of this tRNA, we wanted to determine whether the lack of m6t6A37 induced +1 frameshifting at a similar site. A plasmid (pTHF71) that harbored a hybrid lacZ gene was constructed such that the ribosomes must shift from the zero frame to the +1 frame to obtain a functional β-galactosidase enzyme (18). An ACC-A sequence, which may be a potential +1 frameshifting site for this tRNA, was included within the frameshifting window. This plasmid was then introduced into tsaA+ (GRB1319) and tsaA1 (GRB1320) strains. However, no significant difference in the β-galactosidase specific activities between the tsaA+ and tsaA1 strains was observed (data not shown). Furthermore, the tsaA1 mutation did not increase the frameshifting at a prfB (release factor 2 [RF2]) programmed-frameshift site that was modified to have ACC at the slip site (13). These results thus indicate that the lack of the methyl group of m6t6A does not induce a +1 frameshift at ACC-A and ACC-U sites.

The first step in translation is the selection of the aminoacyl-tRNA to the codon in the A site. An assay that measures the relative rate with which aminoacyl-tRNA selection occurs was developed by Curran and Yarus (14). The assay is based on the programmed +1 frameshifting required to synthesize RF2 (12). We have used this assay to measure the rate of selection of Thr-tRNAGGUThr at ACC and ACU codons in the wild type and in the tsaA1 mutant. However, there was no difference between the tsaA1 mutant and the wild-type control in the rate of Thr-tRNAGGUThr selection (data not shown), suggesting that the m6t6A37 modification did not influence the rate of Thr-tRNAGGUThr selection.

The presence of m6t6A37 improves the reading of the ACC present in the leader mRNA of the thr operon.

The threonine (thrABC) operon of E. coli consists of three genes whose expression is regulated by an attenuator located upstream of the first gene, thrA (Fig. (Fig.3).3). The leader mRNA contains a 21-codon open reading frame in which there are four Ile codons and eight Thr codons (24). Seven of the Thr codons are ACC, which is read by m6t6A37-containing tRNAGGUThr1 and tRNAGGUThr3. The rate with which the ribosome traverses these control Ile and Thr codons determines the level of transcription termination of the leader mRNA (24). We introduced the tsaA1 mutation into a strain containing a transcription fusion in the thrA gene such that the activity of β-galactosidase reflects the level of transcription of the thr operon (35). The data indicate that both in rich (LB) medium and in MOPS-glucose minimal medium the m6t6A37 deficiency of tRNAGGUThr resulted in a twofold derepression, suggesting that the undermodified tRNA is less efficient than the fully modified counterpart in decoding the ACC codon. The measured β-galactosidase activities are as follows: for LB medium, the activity of the wild type was 9.3 ± 1.8 Miller units and that of the tsaA1 mutant was 16 ± 1.0 Miller units; for MOPS-glucose minimal medium, the activity of the wild type was 177 ± 17 Miller units and that of the tsaA1 mutant was 363 ± 22 Miller units. Strains were grown at 37°C to about 3 × 108 cells/ml, and β-galactosidase activity was determined as described in reference 25.

FIG. 3
Features of the regulatory region of the thrABC operon (modified from reference 24). The structure shown is that when the terminator (region 3-4) is formed and when region 1B and region 2 are paired. Region 1A can also pair with region 2, resulting in ...

DISCUSSION

This paper describes the first mutant found to be defective in the synthesis of a t6A derivative; these derivatives are present in the same subset of the tRNAs from all organisms. One such derivative is m6t6A (Fig. (Fig.4),4), which is present only in two tRNA species in E. coli, tRNAGGUThr1 and tRNAGGUThr3. Gene tsaA was localized to min 4.6 of the chromosomal map of E. coli (Fig. (Fig.1)1) and most likely encodes tRNA(m6t6A37)methyltransferase, which catalyzes the addition of the methyl group to the t6A in tRNA. Although lack of this methyl group does not influence the growth rate, the average polypeptide chain elongation rate, and the selection of the Thr-tRNAGGUThr–GTP–EF-Tu ternary complex to the cognate codons ACC and ACU, we observed a twofold derepression of the thr operon (see data in Results). These results suggest that the m6t6A37-modified nucleoside improves the efficiency of tRNAGGUThr1 and tRNAGGUThr3, probably in a step after the aminoacyl-tRNA selection step.

FIG. 4
Structure of m6t6A (N6-methyl-N6-threonylcarbamoyladenosine).

Position 37, which is next to and 3′ of the anticodon, is hypermodified for tRNAs reading codons starting with U (isopentenyl-adenosine derivatives [i6A, ms2i6A, and ms2io6A] or Y base) and A (t6A derivative). It was therefore suggested that the intrinsically weak interaction of the A-U/U-A base pair in the first position of the codon has to be stabilized by a hypermodification at position 37 (19, 29). Their universal occurrence suggests that the t6A derivatives play some essential role in the performance of the tRNA, perhaps in the stabilization of the codon-anticodon interaction by stacking onto the base-paired complex. Bulk tRNA isolated from E. coli starved for threonine and, therefore, deficient for the t6A modifications, does not function normally in in vitro protein synthesis (26). However, we find that the tsaA1 mutation has no effect on the average polypeptide chain elongation rate in vivo. There may be several reasons for the apparent difference between the results of these experiments. First, our mutation does not prevent threonylation, but only the methylation of t6A, and it is possible that the threonyl group provides much of the stabilizing effect. This is consistent with the fact that most of the tRNAs with t6A37 are also not methylated. It will be of interest to isolate and study mutants that fail to threonylate tRNA. Second, the assay may not be sensitive enough to detect minor effects at the relatively small set of codons (codons ACC and ACU) affected by the absence of the 6-methyl group.

To amplify any possible small reduction in the efficiency due to tsaA1, we examined the expression of the thr leader, which contains seven consecutive ACC codons. Here, tsaA1 caused a twofold derepression. This effect may be due either to the summation of a small signal from each codon or to some context effect specific to the cluster. In any case, these results show that the 6-methyl group of m6t6A37 does improve the efficiency of reading the cognate codon ACC. It is possible that this observed derepression could be caused by decreased aminoacylation of tRNAGGUThr1 or tRNAGGUThr3. However, the native tRNAGGUThr3 and the completely unmodified form show similar aminoacylation kinetics (37), suggesting that m6t6A37 does not influence this reaction. Therefore, the derepression of the thr operon-lacZ fusion was likely to be caused by a less-efficient decoding of the ACC codons in the thr leader. Whether this also applies to the other cognate codon (ACU) read by these tRNA species awaits further analysis.

It is useful to compare the effects of tsaA1 to those of the hisT mutation, which prevents the conversion of uridine in the 3′ side of the anticodon arm of primary transcripts to pseudouridine (Ψ). The hisT and tsaA1 mutations have comparable effects on the deattenuation of thr leader constructs. Lynn et al. (24) replaced thr regulatory codon ACC (Thr) with the CAU (His) codon in thr leader mRNA. The CAU codon is translated by tRNAHis, which normally contains Ψ at positions 38 and 39. The hisT mutation derepresses this thr operon allele two- to threefold, i.e., to an extent similar to that observed by us for the tsaA1 mutation. Therefore, the reduction in the translational efficiency of tRNAGGUThr by the m6t6A37 deficiency that was observed may be quantitatively similar to that caused by the absence of Ψ in the anticodon region of tRNAHis. However, unlike the tsaA1 mutation, the hisT mutation strongly reduces growth rate and polypeptide chain elongation rate (31). These substantial differences between the effects of these two mutations on global protein synthesis may be related to large differences in the numbers of affected codons. The tsaA1 mutation affects only two tRNAs and a correspondingly small set of codons. In contrast, nearly all of the tRNAs in E. coli contain Ψ at one or more of positions 38, 39, and 40, and so the hisT mutation affects most tRNAs and codons.

ACKNOWLEDGMENTS

This work was supported by the Swedish Cancer Society (Project 680 to G.R.B.), the Swedish Natural Science Council (project B-BU 2930 to G.R.B.), and by NIH grant GM52643 to J.F.C.

We thank Kerstin Jacobsson and Gunilla Jäger for excellent technical assistance in performing HPLC and mass spectrum analysis; T. Hagervall for plasmid pTHF71; I. Saint-Girons for strain GT527; Hans Lundgren, Jaunius Urbonavicius, and Michael Wikström for critical reading of manuscript; and Mia Bånghagen, Lena Sundberg, and Matthew Marklund for assisting in some experiments.

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