Important role of the amino acid attached to tRNA in formylation and in initiation of protein synthesis in Escherichia coli.

In attempts to convert an elongator tRNA to an initiator tRNA, we previously generated a mutant elongator methionine tRNA carrying an anticodon sequence change from CAU to CUA along with the two features important for activity of Escherichia coli initiator tRNA in initiation. This mutant tRNA (Mi:2 tRNA) was active in initiation in vivo but only when aminoacylated with methionine by overproduction of methionyl-tRNA synthetase. Here we show that the Mi:2 tRNA is normally aminoacylated in vivo with lysine and that the tRNA aminoacylated with lysine is a very poor substrate for formylation compared with the same tRNA aminoacylated with methionine. By introducing further changes at base pairs 4:69 and 5:68 in the acceptor stem of the Mi:2 tRNA to those found in the E. coli initiator tRNA, we show that change of the U4:A69 base pair to G4:C69 and overproduction of lysyl-tRNA synthetase and methionyl-tRNA transformylase results in partial formylation of the mutant tRNA and activity of the formyllysyl-tRNAs in initiation of protein synthesis. Thus, the G4:C69 base pair contributes toward formylation of the tRNA and protein synthesis in E. coli can be initiated with formyllysine. We also discuss the implications of these and other results on recognition of tRNAs by E. coli lysyl-tRNA synthetase and on competition in cells among aminoacyl-tRNA synthetases.

In attempts to convert an elongator tRNA to an initiator tRNA, we previously generated a mutant elongator methionine tRNA carrying an anticodon sequence change from CAU to CUA along with the two features important for activity of Escherichia coli initiator tRNA in initiation. This mutant tRNA (Mi:2 tRNA) was active in initiation in vivo but only when aminoacylated with methionine by overproduction of methionyl-tRNA synthetase. Here we show that the Mi:2 tRNA is normally aminoacylated in vivo with lysine and that the tRNA aminoacylated with lysine is a very poor substrate for formylation compared with the same tRNA aminoacylated with methionine. By introducing further changes at base pairs 4:69 and 5:68 in the acceptor stem of the Mi:2 tRNA to those found in the E. coli initiator tRNA, we show that change of the U4:A69 base pair to G4:C69 and overproduction of lysyl-tRNA synthetase and methionyl-tRNA transformylase results in partial formylation of the mutant tRNA and activity of the formyllysyl-tRNAs in initiation of protein synthesis. Thus, the G4: C69 base pair contributes toward formylation of the tRNA and protein synthesis in E. coli can be initiated with formyllysine. We also discuss the implications of these and other results on recognition of tRNAs by E. coli lysyl-tRNA synthetase and on competition in cells among aminoacyl-tRNA synthetases.
Two species of methionine tRNAs are present in all organisms. The initiator methionine tRNA is used for initiation of protein synthesis, whereas the elongator methionine tRNA is used for insertion of methionine into internal peptidic linkages (1)(2)(3). In eubacteria, mitochondria, and chloroplasts, the initiator tRNA is used as formylmethionyl-tRNA (4) (fMet-tRNA), 1 whereas in the cytoplasm of eukaryotes it is used as methionyl-tRNA (Met-tRNA). In Escherichia coli, formylation of the initiator Met-tRNA is important for initiation of protein synthesis (5)(6)(7). This reaction is catalyzed by the enzyme methionyl-tRNA transformylase (8).
The distinct roles of the two species of methionine tRNAs in protein synthesis are a result of their different structures. In E. coli, the C1xA72 mismatch at the end of the acceptor stem along with G2:C71 and C3:G70 base pairs are important for formylation of the tRNA (9 -11), and the three consecutive G:C base pairs in the anticodon stem are important for binding of the fMet-tRNA to the ribosomal P site (12). However, transplanting these features into the E. coli elongator methionine tRNA also carrying an anticodon sequence change from CAU to CUA did not generate a tRNA that was in itself active in initiation. The tRNA thus generated, Mi:2 tRNA, could initiate protein synthesis only in cells overproducing MetRS (13). The reason for it was that the mutant tRNA was formylated when aminoacylated with methionine but not with the amino acid normally attached to the Mi:2 tRNA. In contrast, transplanting the same features into E. coli glutamine tRNA resulted in a tRNA, Qi:2 tRNA, which was fairly active in initiation (13). A possible explanation of these results is that unlike the Qi:2 tRNA, which is most likely aminoacylated with glutamine, the Mi:2 tRNA is aminoacylated with an amino acid that negatively affects its formylation and/or some subsequent step in initiation of protein synthesis. A further possibility is that E. coli tRNA Gln and tRNA fMet have in common some sequence and/or structural feature that contributes toward initiation and that is lacking in the elongator tRNA Met .
In this paper, we show that the Mi:2 tRNA is aminoacylated with lysine in vivo and that when aminoacylated with lysine, it is a very poor substrate for formylation. We have introduced further changes at base pairs 4:69, 5:68, and both 4:69 and 5:68 in the acceptor stem of the Mi:2 tRNA to G:C base pairs present in E. coli tRNA fMet and in tRNA Gln and studied the effect of these changes on aminoacylation of the tRNA with LysRS and on formylation. We show that the mutant tRNAs with the G4:C69 base pair (Mi:2/4 and Mi:2/4, 5 tRNA) are now better substrates for formylation. Overproduction of LysRS and MTF leads to partial formylation of these mutant tRNAs and the formyllysyl-tRNAs are active in initiation of protein synthesis. These results highlight the role of the amino acid attached to the tRNA in formylation and thereby in initiation of protein synthesis and the role of the G4:C69 base pair in recognition of the tRNA by MTF, specifically in the sequence context of the E. coli tRNA Met . They also show that protein synthesis in E. coli can be initiated with formyllysine.
We have also studied the effects of overproduction of glutaminyl-tRNA synthetase (data not shown), MetRS, and MetRS and MTF on aminoacylation of the mutant tRNAs and on their activities in formylation and initiation. We discuss the implications of these and other results on recognition of tRNAs by LysRS and on competition in cells between glutaminyl-tRNA synthetase, LysRS, and MetRS for tRNAs.

E. coli Strains and
Plasmids-E. coli strain CA274 (HfrHLac-ZamTrpEam) was used as the host strain for analysis of the activity of the various mutant tRNA genes. The tRNA genes were cloned into pRSVpCATam1.2.5 and pRSVpCATam5-6H plasmids as described (14,15). The genes for MetRS, LysRS, and MTF were cloned into pACD (16), a derivative of pACYC184.
Assays for Chloramphenicol Acetyltransferase (CAT) Activity in Extracts-CA274 containing the pRSVpCATam1.2.5 and the pACD vectors were grown at 37°C overnight in 2 ϫ YT medium containing ampicillin (100 mg/ml) and tetracycline (12.5 mg/ml). The overnight culture was diluted 1-20-to 1-200-fold in 2 ϫ YT medium containing ampicillin (50 mg/ml) and tetracycline (8 mg/ml) and was grown for about 4 h at 37°C to A 600 /ml of 0.3-0.5. Cell extracts from 1.2 ml of the fresh culture were used for assays of CAT activity and ␤-lactamase activity (5). CAT activity was normalized to ␤-lactamase activity to eliminate the effect of any possible variations in copy number of the pRSVpCAT plasmid. The relative CAT activity in cells carrying the U35A36 mutant of E. coli initiator tRNA was fixed at 100%.
Purification and Amino-terminal Sequence Analysis of CAT Protein Carrying Six Histidine Residues-CAT protein with six histidine residues was purified by adsorption to nickel-chelate resin (X PRESS SYS-TEM, Invitrogen Inc.) as described (15) except that native conditions were used for binding of the protein and washing of the resin. E. coli CA274 containing pRSVpCATam5-6H/Mi:2 was grown overnight in 1 liter of 2xYT medium at 37°C and harvested by centrifugation at 4°C. The cell pellet was resuspended in 6.5 ml of native binding buffer (20 mM sodium phosphate and 500 mM sodium chloride, pH 7.8) containing 0.1 mg/ml lysozyme and 1 mM phenylmethylsulfonyl fluoride. The cell suspension was incubated on ice for 15 min, frozen and thawed three times, and centrifuged for 10 min at 4°C. The supernatant was collected and mixed with nickel-chelate resin (3.6 ml) that was equilibriated with native binding buffer, pH 7.8. After incubation at 4°C for 2 h with constant mixing, the resin was washed with native washing buffer (same as binding buffer except that pH was 6.0) until an A 280 /ml of 0.02 was obtained. The resin was further washed with native pH elution buffer (same as binding buffer, except that pH was 4.0) or native imidazole elution buffer (like native buffer except 0.3 M imidazole was included, pH 6.0). The bound proteins were eluted in Laemmli sample buffer containing 2% SDS, 0.062 M Tris-HCl, pH 6.8, and 14.4 mM ␤-mercaptoethanol. Five fractions (1 ml each) were collected, and the fractions containing CAT protein were identified by immunoblot analysis using anti-CAT antibody and concentrated by methanol precipitation (17). The concentrated proteins were separated on a 12% SDSpolyacrylamide gel, and the proteins were electroblotted onto a polyvinylidene difluoride membrane (Immobilon P; pore size, 0.45 M; Millipore) and stained briefly (5 min) with Coomassie Blue. One of the lanes containing ϳ1% of total protein applied was sliced off and used for immunodetection of CAT protein using anti CAT antibodies (5). The CAT protein band on the Coomassie-stained membrane was excised and used for sequencing (18).
Isolation and Northern Blot Analysis of tRNAs-Cells expressing the mutant tRNAs (approximately 3 ml) were grown to an A 600 /ml of 0.3-0.5 (see above), pelleted, resuspended in 0.3 M sodium acetate, pH 4.5, and 1 mM EDTA, and extracted with an equal volume of phenol equilibriated with the same buffer (19). The aqueous layer was transferred to another tube and extracted once more with phenol. The final aqueous layer was then mixed with 3 volumes of ethanol and left on dry ice for 30 min to precipitate the tRNAs. The tRNAs were dissolved in 10 mM sodium acetate, pH 4.5, and 1 mM EDTA. The tRNAs were separated by electrophoresis on an acid-urea gel (19) and electroblotted onto Nytran membrane, and the mutant tRNAs were detected by hybridization to a 32 P-labeled oligonucleotide probe (5Ј-CCAGGGTTTAGAGCCCTGTG-3Ј) complementary to nucleotides 25-44 of the mutant elongator methionine tRNAs described in this work.

RESULTS
The Mi:2 tRNA Is Aminoacylated with Lysine in Vivo-In attempts to switch an elongator tRNA to an initiator tRNA, we had previously transplanted into E. coli tRNA Met several of the features important for activity of the E. coli initiator tRNA in initiation. These mutations were coupled to the anticodon sequence change from CAU to CUA to allow assessment of the initiation activity of the mutant tRNAs in vivo (13). Fig. 1 shows a clover leaf structure of one of the mutant tRNAs, the Mi:2 tRNA. This tRNA was active in initiation in vivo but only in cells overproducing MetRS. This is because the mutant tRNA was formylated when aminoacylated with methionine but not with the amino acid normally attached to the Mi:2 tRNA. In cells not overproducing MetRS, the Mi:2 tRNA was partially aminoacylated but not formylated (13). Although the identity of the amino acid attached to the Mi:2 tRNA was not established then, we considered the possibility that it was lysine or glutamine.
To determine the identity of the amino acid attached to the Mi:2 tRNA, we have taken advantage of the fact that although this mutant tRNA with a C1xA72 mismatch at the end of the acceptor stem has a much reduced activity in elongation, it can act as a weak suppressor of amber codons (20). 2 The Mi:2 tRNA was, therefore, cloned into the plasmid vector (CATam5-6H) carrying a mutant chloramphenicol acetyltransferase reporter gene with a UAG amber codon at position 5 followed by six histidine codons. The CAT protein made was purified by affinity chromatography and subjected to amino-terminal sequence analysis (18). Results in Fig. 2 show that the amino acid inserted at the site of the UAG codon by the Mi:2 tRNA is lysine.
Introduction of Additional Mutations into the Mi:2 tRNA Gene-In contrast to the Mi:2 tRNA, which is not formylated in cells unless MetRS is overproduced, the Qi:2 tRNA derived from tRNA Gln is essentially all formylated (13). Both mutant tRNAs contain the same nucleotides at positions 1 and 72 and base pairs 2:71 and 3:70 in the acceptor stem, which are important for formylation (9). The difference between the two tRNAs in the acceptor stem is the absence in the Mi:2 tRNA and the presence in the Qi:2 tRNA of base pairs G4:C69 and G5:C68 found in the E. coli initiator tRNA. Other studies have also implicated a potential role for G4:C69 and G5:C68 base pairs on formylation (9)  observations, we introduced these G:C base pairs into the Mi:2 tRNA gene to investigate whether these mutant tRNAs would now be better substrates for formylation and hence be active in initiation without requiring the overproduction of MetRS. The new mutant tRNAs are designated as Mi:2/4, Mi:2/5, and Mi: 2/4,5 tRNAs (Fig. 1).
Activity of Mi:2, Mi:2/4, Mi:2/5, and Mi:2/4,5 tRNAs in Initiation in Cells Overproducing MTF, LysRS, and Both MTF and LysRS-Previous work has shown that activity of mutant initiator tRNAs that are defective in initiation due to a block in formylation can be rescued by overproduction of the formylating enzyme MTF (16,20). Therefore, we investigated the effect of overproduction of MTF, LysRS, and both MTF and LysRS on activity of the mutant tRNAs in initiation from a UAG initiation codon (5,13,14). Fig. 3 shows the vectors used to coexpress the CAT reporter gene and the mutant tRNAs (pRSVpCA-Tam1.2.5) and to overproduce the various enzymes (pACD). Table I shows the results. Overproduction of MTF alone leads to a small increase in CAT activity with all of the mutant tRNAs. Overproduction of LysRS alone has no effect on CAT activity with any of the mutant tRNAs tested. However, overproduction of MTF and LysRS results in a substantial increase in CAT activity in cells expressing the Mi:2/4 and Mi:2/4,5 tRNAs but not in those expressing the Mi:2 and the Mi:2/5 tRNAs. Immunoblot analysis of extracts of cells expressing the mutant tRNAs and overproducing MTF and LysRS (Fig. 4) also confirms these results. A band corresponding to CAT protein is seen in extracts of cells carrying the Mi:2/4 and Mi:2/4,5 tRNAs but not the Mi:2 and Mi:2/5 tRNAs. All of the extracts contain ␤-lactamase, another protein encoded by the same pRSVpCA- In cells not overproducing any of the enzymes (vector alone, lane 1 of Fig. 5, A-D), the mutant tRNAs are aminoacylated with lysine to varying degrees. When LysRS is overproduced, all the mutant tRNAs are essentially quantitatively aminoacylated with lysine (lanes 3 of Fig. 5, A-D). However, the Lys-tRNAs are not formylated. When both MTF and LysRS are overproduced, the Lys-tRNAs corresponding to the Mi:2/4 and Mi:2/4,5 tRNAs are now partially formylated. These are also the two tRNAs that showed clear activity in initiation in vivo under these conditions. There was no formylation of Lys-tRNAs corresponding to the Mi:2 and Mi:2/5 tRNAs in spite of the fact that the tRNAs are essentially quantitatively aminoacylated. These results suggest that the presence of a G4:C69 base pair makes the Lys-tRNAs better substrates for MTF, whereas the presence of a G5:C68 base pair has no such effect or a marginal effect at the most.
Although all of the above mutant tRNAs are aminoacylated with lysine in cells not overproducing any of the enzymes (lanes 1, Fig. 5, A-D), there are differences in the extent of aminoacylation among the mutant tRNAs. The relative intensity of the Lys-tRNA band compared with the uncharged tRNA band is clearly higher for the Mi:2/5 and Mi:2/4,5 tRNAs than for the other tRNAs. Table II Table III shows the results on activity of the mutant tRNAs in initiation. The data on the effect of overproduction of MTF alone are the same as described above (Table I)

FIG. 4. Immunoblot analysis of CAT and ␤-lactamase levels in extracts from E. coli CA274 transformants carrying the various mutant tRNA genes and overproducing LysRS and MTF.
Cell extracts were prepared from CA274 transformants carrying the different pRSVpCATam1.2.5 vectors (Fig. 3) with the mutant tRNAs as indicated and the pACD vector containing the genes for LysRS and MTF. Immunoblot analysis was carried out as described elsewhere (16).  in CAT activity of about 3-fold. This is most likely because more of the Mi:2 tRNA that is aminoacylated with methionine is now formylated (Fig. 6A, compare lanes 3 and 4). With the Mi:2/4 tRNA, CAT activity in cells carrying this tRNA and overproducing MetRS alone is about 2-fold higher than in cells carrying the Mi:2 tRNA; however, there is no further increase upon overproduction of MTF. This is consistent with the conclusion above that the presence of a G4:C69 base pair makes the Mi:2/4 tRNA a better substrate for MTF, such that essentially all of the tRNA that is aminoacylated with methionine in cells overproducing MetRS is now formylated (Fig. 6C, compare lanes 3  and 4). With the Mi:2/5 tRNA, the results are essentially the same as with the Mi:2 tRNA. With the Mi:2/4,5 tRNA, the effect of overproduction of MTF and MetRS or MetRS alone basically mirror those seen with the Mi:2/4 tRNA, except that CAT activities in cells carrying the Mi:2/4,5 tRNA is about half those with the Mi:2/4 tRNA. This is probably because this tRNA is the best substrate for LysRS among the four tRNAs tested (Table II). Consequently, in cells overproducing MetRS, less of the Mi:2/4,5 tRNA gets aminoacylated with methionine and subsequently formylated than the Mi:2/4 tRNA (Fig. 6, C and D, compare the relative intensities of the band corresponding to fMet-tRNA to bands corresponding to formyllysyl-tRNAs and Lys-tRNA combined in lanes 3 and 4 of C to lanes 3 and 4 of D, respectively).

Recognition of Aminoacyl-tRNAs by MTF: Role of the Amino
Acid and tRNA Sequence-This study has provided a clear indication of the important role of the amino acid attached to the tRNA in formylation and in initiation of protein synthesis in E. coli. We have shown that the Mi:2 mutant tRNA is aminoacylated with lysine in vivo and that the tRNA charged with lysine is an extremely poor substrate for formylation. For example, in cells overproducing LysRS, the mutant tRNA is essentially all aminoacylated with lysine, but the Lys-tRNA is not formylated (lane 3 of Fig. 5A and Table I). In contrast, in cells overproducing MetRS, a part of the tRNA is aminoacyl-ated with methionine, and the Met-tRNA is now substantially formylated (lane 3 of Fig. 6A; see also Fig. 4 of Ref. 13). These results show that MTF has a very strong preference for methionine over lysine. Previous work on a mutant of E. coli initiator tRNA aminoacylated with glutamine (9,22) and the initiator tRNA misaminoacylated with phenylalanine and valine (23) suggested a certain hierarchy in the preference of MTF for amino acids attached to tRNA in the following order: MetϾGlnϾPheϾVal. The current work allows us to add Lys to this list and indicates that it is among the least preferred of the amino acids for MTF.
The amino acid moiety also plays a role in at least one later step in initiation of protein synthesis subsequent to formylation of the tRNA. Previous studies have shown that the U35A36 mutant of E. coli initiator tRNA is a better initiator when it carries fMet than when it carries fGln (21). This suggests that some component of the translational machinery that interacts directly with the initiator tRNA prefers fMet over fGln. Because the initiation factor IF2 is known to interact with the acceptor stem of the tRNA (24) and requires an N-acylated amino acid for binding (25), the affinity of IF2 for the formylaminoacid moiety could be another factor that determines the activity of a tRNA in initiation. Some evidence for this possibility comes from the finding that activity of the U35A36 mutant initiator tRNA carrying fGln is increased greatly upon overproduction of IF2 (16). Thus, the absolute conservation of methionine as the initiating amino acid in all organisms could be due to the fact that the amino acid is inspected at two distinct steps in the initiation pathway with methionine being the most preferred amino acid at each of these steps. In eukaryotes where the initiator tRNA is not formylated, there are indications that the initiation factor eIF2 may have an absolute requirement for methionine attached to the initiator tRNA (26). 3 The introduction of further changes in base pairs 4:69 and 5:68 in the acceptor stem of the Mi:2 tRNA to G:C base pairs found at these positions in the E. coli initiator tRNA has shown that the presence of the G4:C69 base pair makes the tRNA a better substrate for MTF. Thus, in cells overproducing LysRS and MTF, the Mi:2 tRNA is aminoacylated but is not formylated. Consequently, it has very little activity in initiation. In contrast, a substantial fraction of the Mi:2/4 and Mi:2/4,5 tRNAs carrying the G4:C69 base pair is now formylated in vivo (lane 4 of Fig. 5, C and D), and these tRNAs show significant activity in initiation. The Mi:2/5 tRNA behaves essentially similarly to the Mi:2 tRNA, suggesting that the presence of the G5:C68 base pair does not have the same effect as the G4:C69 base pair; there is no detectable formylation of Mi:2/5 tRNA in cells overproducing LysRS and MTF (lane 4 of Fig. 5B). Besides

TABLE II
The percentage of aminoacylation with lysine of Mi:2 tRNA and its derivatives The percentage of aminoacylation, based on phosphorimager analysis of a Northern blot, is defined as counts in the lysyl-tRNA band divided by counts in the lysyl-tRNA plus the tRNA band X100. Intensity in the bands was measured using the Image Quant Software.  a The CAT activities are normalized to that of tRNA fMet (CUA), which is set as 100%. b The data are the same as in Table I, used here for comparison. c Average of three separate assays on three individual extracts. All the other numbers are average of two to six individual assays. d This strain has a tendency to lose the pRSVpCATam1.2.5 plasmid carrying the CAT reporter and the tRNA gene at a high frequency. Only a fraction of the cells from an overnight culture grown in the presence of ampicillin and tetracycline still contain the pRSV plasmid. Because the cell density of the fresh culture from an inoculum of the overnight culture was less than that of the other cultures at the time of harvesting, two to two and a half times as much of the fresh culture of this strain was used for preparation of the cell extracts compared to the other cultures (see "Materials and Methods"). confirming previous conclusions (5, 6) on the importance of formylation of tRNA in initiation of protein synthesis, these results show that protein synthesis in E. coli can be initiated with formyllysine. Thus, besides methionine, the list of amino acids shown to initiate protein synthesis in E. coli now includes Lys in addition to Gln, Ile, Phe, Val, Cys, Trp, and Tyr (5,14,(27)(28)(29).
The clear effect in vivo of the presence of the G4:C69 base pair in formylation of the Mi:2/4 and Mi:2/4,5 tRNAs, derived from E. coli elongator methionine tRNA, is in agreement with previous conclusions based on in vitro experiments that importance of the G4:C69 base pair on formylation depends on the tRNA context. Mutation of G4:C69 base pair to C:G base pair in the E. coli initiator tRNA sequence background has a relatively small effect lowering V max /K m in formylation by a factor of about 4.7 (9). In contrast, mutation of G4:C69 in an elongator methionine tRNA background has a much more severe effect, lowering V max /K m by a factor of about 100 (10).
It is important to note that in spite of the observed formylation of the Mi:2/4 and Mi:2/4,5 tRNAs, these tRNAs are still very poor substrates for formylation when aminoacylated with lysine. Formylation of these tRNAs is partial and detectable only in cells overproducing MTF. In contrast, essentially all of the tRNAs aminoacylated with methionine is formylated in cells overproducing MetRS and MTF. These results further highlight the important role of the amino acid discussed above in formylation.
Recognition of tRNAs by LysRS-In attempts to generate amber suppressors derived from different tRNAs, Abelson, Miller and colleagues (30) found that these suppressors could be divided into three classes: those that inserted the predicted amino acid, those that inserted predominantly glutamine, and those that inserted predominantly lysine. It is not known why amber suppressors derived from some tRNAs such as the E. coli initiator tRNA or tRNA 1 Ile are aminoacylated with glutamine, whereas those derived from the elongator methionine tRNA and tRNA 2 Ile are aminoacylated with lysine. One possible correlation, based on genetic and biochemical studies of tRNA mutants (22,(31)(32)(33)(34)) and x-ray crystallographic analysis of the E. coli glutaminyl-tRNA synthetase⅐tRNA Gln complex (35), is that tRNAs aminoacylated with glutaminyl-tRNA synthetase either have a weak base pair between nucleotides 1 and 72 that can be easily disrupted by the enzyme or have G73 in the discriminator position. G73 stabilizes a form of the acceptor stem structure in which the 1:72 base pair is disrupted and the tRNA-CCA end is curled back toward the anticodon. However, an anticodon mutant of E. coli asparagine tRNA that has a weak U1:A72 base pair and G73 is aminoacylated with lysine and not with glutamine (36). Also, although the aminoacylation of the amber suppressor derived from the elongator methionine tRNA, which has a strong G1:C72 base pair, with lysine rather than glutamine could be rationalized on this basis (30), the current finding that the Mi:2 tRNA with a C1xA72 mismatch is also aminoacylated with lysine is not easily explained. It is possible that the Mi:2 tRNA and tRNA Asn contains other determinants for LysRS that predominate over the above features. Alternatively, they have features that act as negative determinants toward E. coli glutaminyl-tRNA synthetase (37). The finding that the Mi:2 tRNA with a C1xA72 mismatch is aminoacylated with lysine in vivo suggests that the presence of a G1:C72 base pair found in tRNA Lys is not essential for its aminoacylation by LysRS.
The Mi:2/4,5 tRNA is also aminoacylated with lysine in vivo. This tRNA has the first 5 base pairs in the acceptor stem, the last 3 base pairs in the anticodon stem, and the entire sequence of the anticodon loop identical to the U35A36 mutant of E. coli initiator tRNA. The latter tRNA is aminoacylated with glutamine in vitro and in vivo (14,22,33). These results suggest that the sequence and/or structural features in the Mi:2/4,5 tRNA that determine whether the tRNA is aminoacylated in vivo with glutamine or lysine probably reside outside of these regions.
It is interesting that changing the fourth and in particular the fifth base pair in the acceptor stem of the Mi:2 tRNA from U4:A69 and A5:U68 to G:C base pairs makes these tRNAs better substrates for LysRS in vivo. The lysine tRNA of E. coli does not have G:C base pairs at these positions, in fact it has U4:A69 and C5:G68 (38). It is possible that these changes in the Mi:2 tRNA affect the acceptor stem structure in such a way that it fits better into the catalytic pocket of LysRS. It is also possible that LysRS interacts directly with the fifth base pair of lysine tRNA. The G5:C68 base pair of the Mi:2/5 tRNA could provide some of the same contacts as that provided by a C5:G68 base pair (39). In this regard, it may be relevant that an amber suppressor derived from E. coli tRNA Arg with a C5:G68 base pair is partially aminoacylated in vivo with lysine, whereas that derived from another species of tRNA Arg with a U5:G68 base pair is instead partially aminoacylated with glutamine (40,41).
Finally, whether a mutant tRNA is aminoacylated in vivo with lysine, glutamine, methionine, or other amino acids depends upon competition in cells between the various amino- acyl-tRNA synthetases and the tRNA in question (21,(42)(43)(44). Because the lysine side chain has a positive charge, a tRNA aminoacylated with lysine is easily differentiated from the same tRNA aminoacylated with glutamine, methionine, and most other amino acids by its slower mobility on polyacrylamide gels run under acidic conditions (13,19)