(Received for publication, October 2, 1995)
From the
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), ()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, 10, 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 and tRNA
have in common
some sequence and/or structural feature that contributes toward
initiation and that is lacking in the elongator tRNA
.
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 and in tRNA
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
. 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.
Figure 1: The nucleotide sequence in cloverleaf form of the Mi:2 mutant tRNA. This tRNA, derived from E. coli elongator methionine tRNA, has the following mutations: C1xA72/G29G30G31:C39C40C41, in addition to the anticodon sequence change from CAU to CUA. The Mi:2/4, Mi:2/5, and Mi:2/4,5 tRNAs are derived from the Mi:2 tRNA by further mutations of the fourth, the fifth, or both the fourth and the fifth base pairs in the acceptor stem (indicated by arrows). X stands for 3-amino carboxypropyl uridine present in the wild type elongator methionine tRNA. Whether this modification is present in the mutant tRNAs studied here is not known.
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) . ()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. 2show that the
amino acid inserted at the site of the UAG codon by the Mi:2 tRNA is
lysine.
Figure 2: Identification of amino acid inserted by the Mi:2 tRNA as lysine. A, the scheme used. B, the yield of various amino acids in picomoles plotted against the cycle number during Edman degradation of the CAT reporter protein.
Figure 3: The vectors used to coexpress the CAT reporter gene and tRNA mutants (left) and LysRS, MetRS, or MTF (right) in E. coli. The gene for MTF (designated FMT by Guillon et al. in (6) ) is on a DNA fragment that contains the gene for a peptide deformylase upstream of the FMT gene. These genes are part of an operon in E. coli and are transcribed as a dicistronic mRNA(45) . The gene for MetRS and the lysU gene for LysRS (46) was cloned into the EcoRI site.
Figure 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) .
Figure 5: Northern blot analysis of the various mutant tRNAs: Mi:2 (A), Mi:2/5 (B), Mi:2/4 (C), and Mi:2/4,5 (D) isolated from CA274 transformants overproducing MTF, LysRS, or both LysRS and MTF. The location of uncharged tRNA on the blot was confirmed by base hydrolysis of one of the tRNA preparations prior to electrophoresis (data not shown).
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 2provides a quantitative estimate of aminoacylation (19) of the four tRNAs with lysine using a PhosphorImager analysis of a Northern blot on a different batch of tRNAs. The results show that the presence of G4:C69 and G5:C68 base pairs on the Mi:2 tRNA make the tRNAs better substrate for LysRS, the effect of the G5:C68 base pair being more pronounced than that of the G4:C69 base pair.
Figure 6: Northern blot analysis of the various mutant tRNAs: Mi:2 (A), Mi:2/5 (B), Mi:2/4 (C), and Mi:2/4,5 (D) isolated from CA274 transformants overproducing MTF, MetRS, or both MetRS and MTF. Not indicated on the figure is the location of Met-tRNA, which migrates between fLys-tRNA and fMet-tRNA (see Fig. 4of (13) ).
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) . ()
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 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/K
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
/K
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.
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 with a C5:G68 base pair is partially
aminoacylated in vivo with lysine, whereas that derived from
another species of tRNA
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 aminoacyl-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) (lanes 3 and 4 of Fig. 6, A-D; see also Fig. 4of (13) ). Inspection of autoradiograms corresponding to Fig. 5exposed for long periods shows that none of the mutant tRNAs are aminoacylated with any amino acid other than lysine. Furthermore, whereas overproduction of LysRS leads to essentially complete aminoacylation of each of the mutant tRNAs with lysine (lane 3 of Fig. 5, A-D), overproduction of glutaminyl-tRNA synthetase (data not shown, see Fig. 4of (13) ) or of MetRS (lane 3 of Fig. 6, A-D and Fig. 4of (13) ) still leaves most of the tRNA in the uncharged form. These data indicate that Mi:2, Mi:2/4, Mi:2/5, and Mi:2/4,5 tRNAs are all better substrates for LysRS than they are for either glutaminyl-tRNA synthetase or for MetRS.