Common location of determinants in initiator transfer RNAs for initiator-elongator discrimination in bacteria and in eukaryotes

Initiator tRNAs are used exclusively for initiation of protein synthesis and not for elongation. We show that both Escherichia coli and eukaryotic initiator tRNAs have negative determinants, at the same positions, that block their activity in elongation. The primary negative determinant in E. coli initiator tRNA is the C1xA72 mismatch at the end of the acceptor stem. The primary negative determinant in eukaryotic initiator tRNAs is located in the TPsiC stem, whereas a secondary negative determinant is the A1:U72 base pair at the end of the acceptor stem. Here we show that E. coli initiator tRNA also has a secondary negative determinant for elongation and that it is the U50.G64 wobble base pair, located at the same position in the TPsiC stem as the primary negative determinant in eukaryotic initiator tRNAs. Mutation of the U50.G64 wobble base pair to C50:G64 or U50:A64 base pairs increases the in vivo amber suppressor activity of initiator tRNA mutants that have changes in the acceptor stem and in the anticodon sequence necessary for amber suppressor activity. Binding assays of the mutant aminoacyl-tRNAs carrying the C50 and A64 changes to the elongation factor EF-Tu.GTP show marginally higher affinity of the C50 and A64 mutant tRNAs and increased stability of the EF-Tu.GTP. aminoacyl-tRNA ternary complexes. Other results show a large effect of the amino acid attached to a tRNA, glutamine versus methionine, on the binding affinity toward EF-Tu.GTP and on the stability of the EF-Tu.GTP.aminoacyl-tRNA ternary complex.     

The fate of the initiator tRNAs is sensitive to the critical balance between interacting proteins

Formylation of the initiator tRNA is essential for normal growth of Escherichia coli. The initiator tRNA containing the U35A36 mutation (CUA anticodon) initiates from UAG codon. However, an additional mutation at position 72 (72A --> G) renders the tRNA (G72/U35A36) inactive in initiation because it is defective in formylation. In this study, we isolated U1G72/U35A36 tRNA containing a wobble base pair at 1-72 positions as an intragenic suppressor of the G72 mutation. The U1G72/U35A36 tRNA is formylated and participates in initiation. More importantly, we show that the mismatch at 1-72 positions of the initiator tRNA, which was thus far thought to be the hallmark of the resistance of this tRNA against peptidyl-tRNA hydrolase (PTH), is not sufficient. The amino acid attached to the initiator tRNA is also important in conferring protection against PTH. Further, we show that the relative levels of PTH and IF2 influence the path adopted by the initiator tRNAs in protein synthesis. These findings provide an important clue to understand the dual function of the single tRNA(Met) in initiation and elongation, in the mitochondria of various organisms.     

Suppression of amber codons in vivo as evidence that mutants derived from Escherichia coli initiator tRNA can act at the step of elongation in protein synthesis

The absence of a Watson-Crick base pair at the end of the amino acid acceptor stem is one of the features which distinguishes prokaryotic initiator tRNAs as a class from all other tRNAs. We show that this structural feature prevents Escherichia coli initiator tRNA from acting as an elongator in protein synthesis in vivo. We generated a mutant of E. coli initiator tRNA in which the anticodon sequence is changed from CAU to CUA (the T35A36 mutant). This mutant tRNA has the potential to read the amber termination codon UAG. We then coupled this mutation to others which change the C1.A72 mismatch at the end of the acceptor stem to either a U1:A72 base pair (T1 mutant) or a C1:G72 base pair (G72 mutant). Transformation of E. coli CA274 (HfrC Su- lacZ125am trpEam) with multicopy plasmids carrying the mutant initiator tRNA genes show that mutant tRNAs carrying changes in both the anticodon sequence and the acceptor stem suppress amber codons in vivo, whereas mutant tRNA with changes in the anticodon sequence alone does not. Mutant tRNAs with the above anticodon sequence change are aminoacylated with glutamine in vitro. Measurement of kinetic parameters for aminoacylation by E. coli glutaminyl-tRNA synthetase show that both the nature of the base pair at the end of the acceptor stem and the presence or absence of a base pair at this position can affect aminoacylation kinetics. We discuss the implications of this result on recognition of tRNAs by E. coli glutaminyl-tRNA synthetase.     

Binding of tRNA in different functional states to Escherichia coli ribosomes as measured by velocity sedimentation

The binding of initiator and elongator tRNAs to 70-S ribosomes and the 30-S subunits was followed by velocity sedimentation in the analytical ultracentrifuge. fMet-tRNAfMet binds to A-U-G-programmed 30-S subunits, but not to free or misprogrammed particles. Both the formylmethione residue and the initiation factors increase the stability of the 30-S x A-U-G x fMet-tRNAfMet complex. fMet-tRNAfMet is bound only to the P site of the 70-S ribosome even in the absence of A-U-G. Two copies of tRNAPhe or Phe-tRNAPhe are bound to the ribosome with similar affinity. In contrast to a recent report [Rheinberger et al. (1981) Proc. Natl Acad. Sci. USA, 78, 5310-5314], it is shown that three copies of tRNA cannot be bound simultaneously to the ribosome with binding constants higher than 2 x 10(4) M-1. Phe-tRNAPhe when present as the ternary complex Phe-tRNAPhe. EF-Tu x guanosine 5'-[beta,gamma-methylene]triphosphate binds exclusively to the A site. The peptidyl-tRNA analogue, acetylphenylalanine-tRNA, can occupy both ribosomal centers, albeit with a more than tenfold higher affinity for the P site. The thermodynamic data obtained under equilibrium conditions confirm the present view of two tRNA binding sites on the ribosome. The association constants determined are discussed in relation to the mechanism of ribosomal protein synthesis.     

Adaptation to tRNA acceptor stem structure by flexible adjustment in the catalytic domain of class I tRNA synthetases

Class I aminoacyl-tRNA synthetases (aaRSs) use a Rossmann-fold domain to catalyze the synthesis of aminoacyl-tRNAs required for decoding genetic information. While the Rossmann-fold domain is conserved in evolution, the acceptor stem near the aminoacylation site varies among tRNA substrates, raising the question of how the conserved protein fold adapts to RNA sequence variations. Of interest is the existence of an unpaired C-A mismatch at the 1-72 position unique to bacterial initiator tRNA(fMet) and absent from elongator tRNAs. Here we show that the class I methionyl-tRNA synthetase (MetRS) of Escherichia coli and its close structural homolog cysteinyl-tRNA synthetase (CysRS) display distinct patterns of recognition of the 1-72 base pair. While the structural homology of the two enzymes in the Rossmann-fold domain is manifested in a common burst feature of aminoacylation kinetics, CysRS discriminates against unpaired 1-72, whereas MetRS lacks such discrimination. A structure-based alignment of the Rossmann fold identifies the insertion of an α-helical motif, specific to CysRS but absent from MetRS, which docks on 1-72 and may discriminate against mismatches. Indeed, substitutions of the CysRS helical motif abolish the discrimination against unpaired 1-72. Additional structural alignments reveal that with the exception of MetRS, class I tRNA synthetases contain a structural motif that docks on 1-72. This work demonstrates that by flexible insertion of a structural motif to dock on 1-72, the catalytic domain of class I tRNA synthetases can acquire structural plasticity to adapt to changes at the end of the tRNA acceptor stem.     

The crystal structure of Cys-tRNACys-EF-Tu-GDPNP reveals general and specific features in the ternary complex and in tRNA.

BACKGROUND:. The translation elongation factor EF-Tu in its GTP-bound state forms a ternary complex with any aminoacylated tRNA (aa-tRNA), except initiator tRNA and selenocysteinyl-tRNA. This complex delivers aa-tRNA to the ribosomal A site during the elongation cycle of translation. The crystal structure of the yeast Phe-tRNAPhe ternary complex with Thermus aquaticus EF-Tu-GDPNP (Phe-TC) has previously been determined as one representative of this general yet highly discriminating complex formation. RESULTS: The ternary complex of Escherichia coli Cys-tRNACys and T. aquaticus EF-Tu-GDPNP (Cys-TC) has been solved and refined at 2.6 degrees resolution. Conserved and variable features of the aa-tRNA recognition and binding by EF-Tu-GTP have been revealed by comparison with the Phe-TC structure. New tertiary interactions are observed in the tRNACys structure. A 'kissing complex' is observed in the very close crystal packing arrangement. CONCLUSIONS: The recognition of Cys-tRNACys by EF-Tu-GDPNP is restricted to the aa-tRNA motif previously identified in Phe-TC and consists of the aminoacylated 3' end, the phosphorylated 5' end and one side of the acceptor stem and T stem. The aminoacyl bond is recognized somewhat differently, yet by the same primary motif in EF-Tu, which suggests that EF-Tu adapts to subtle variations in this moiety among all aa-tRNAs. New tertiary interactions revealed by the Cys-tRNACys structure, such as a protonated C16:C59 pyrimidine pair, a G15:G48 'Levitt pair' and an s4U8:A14:A46 base triple add to the generic understanding of tRNA structure from sequence. The structure of the 'kissing complex' shows a quasicontinuous helix with a distinct shape determined by the number of base pairs.     

Role of the 1-72 base pair in tRNAs for the activity of Escherichia coli peptidyl-tRNA hydrolase.

Previous work by Schulman and Pelka (1975) J. Biol. Chem. 250, 542-547, indicated that the absence of a pairing between the bases 1 and 72 in initiator tRNA(fMet) explained the relatively small activity of peptidyl-tRNA hydrolase towards N-acetyl-methionyl-tRNA(fMet). In the present study, the structural requirements for the sensitivity of an N-acetyl-aminoacyl-tRNA to Escherichia coli peptidyl-tRNA hydrolase activity have been further investigated. Ten derivatives of tRNA(fMet) with various combinations of bases at positions 1 and 72 in the acceptor stem have been produced, aminoacylated and chemically acetylated. The release of the aminoacyl moiety from these tRNA derivatives was assayed in the presence of peptidyl-tRNA hydrolase purified from an overproducing strain. tRNA(fMet) derivatives with either C1A72, C1C72, U1G72, U1C72 or A1C72 behaved as poor substrates of the enzyme, as compared to those with C1G72, U1A72, G1C72, A1U72 or G1U72. With the exception of U1G72, it could be therefore concluded that the relative resistance of tRNA(fMet) to peptidyl-tRNA hydrolase did not depend on a particular combination of nucleotides at positions 1 and 72, but rather reflected the absence of a base pairing at these positions. In a second series of experiments, the unpairing of the 1 and 72 bases, created with C-A or A-C bases, instead of G-C in methionyl-tRNA(mMet) or in valyl-tRNA(Val1), was shown to markedly decrease the rate of hydrolysis catalysed by peptidyl-tRNA hydrolase. Altogether, the data indicate that the stability of the 1-72 pair governs the degree of sensitivity of a peptidyl-tRNA to peptidyl-tRNA hydrolase.     

Crystal structure of methionyl-tRNAfMet transformylase complexed with the initiator formyl-methionyl-tRNAfMet.

The crystal structure of Escherichia coli methionyl-tRNAfMet transformylase complexed with formyl-methionyl-tRNAfMet was solved at 2.8 A resolution. The formylation reaction catalyzed by this enzyme irreversibly commits methionyl-tRNAfMet to initiation of translation in eubacteria. In the three-dimensional model, the methionyl-tRNAfMet formyltransferase fills in the inside of the L-shaped tRNA molecule on the D-stem side. The anticodon stem and loop are away from the protein. An enzyme loop is wedged in the major groove of the acceptor helix. As a result, the C1-A72 mismatch characteristic of the initiator tRNA is split and the 3' arm bends inside the active centre. This recognition mechanism is markedly distinct from that of elongation factor Tu, which binds the acceptor arm of aminoacylated elongator tRNAs on the T-stem side.     

The ternary complex of aminoacylated tRNA and EF-Tu-GTP. Recognition of a bond and a fold

The refined crystal structure of the ternary complex of yeast Phe-tRNA^Phe, Thermus aquaticus elongation factor EF-Tu and the non-hydrolyzable GTP analog, GDPNP, revelas many details of the EF-Tu recognition of aminoacylated tRNA (aa-tRNA). EF-Tu-GTP recognizes the aminoacyl bond and one side of the backbone fold of the acceptor helix and has a high affinity for all ordinary elongator aa-tRNAs by binding to this aa-tRNA motif. Yet, the binding of deacylated tRNA, initiator tRNA, and selenocysteine-specific tRNA (tRNA^Sec) is effectively discriminated against. Subtle rearrangements of the binding pocket may occur to optimize the fit to any side chain of the aminoacyl group and interactions with EF-Tu stabilize the 3′-aminoacyl isomer of aa-tRNA. A general complementarity is observed in the location of the binding sites in tRNA for synthetases and for EF-Tu. The complex formation is highly specific for the GTP-bound conformation of EF-Tu, which can explain the effects of various mutants.     

Interaction between initiator Met-tRNAfMet and elongation factor EF-Tu from E. coli

It has recently been shown that the non-formylated initiator Met-tRNAfMet from E. coli can form a stable ternary complex with the elongation factor EF-Tu and GTP. Using the protection of EF-Tu:GTP against spontaneous hydrolysis of the aminoacylester bond of Met-tRNAfMet, we confirm these results, and show that the protection is specific for the non-formylated form of the initiator tRNA. The ternary complex Met-tRNAfMet:EF-Tu:GTP can be isolated by column chromatography in a way similar to that demonstrated previously with EF-Tu complexed to the elongator Met-tRNAmMet. 32P-labeled Met-tRNAfMet within the ternary complex was analyzed by the footprinting technique. The pattern of initiator tRNA protection by EF-Tu against ribonuclease digestion is not significantly different from the one found previously for elongator tRNAs. These results lead us to suggest that the initiator tRNAfMet, under growth conditions which do not permit formylation, may to some extent function as an elongator tRNA.     

Recognition of the initiator tRNA by the Pseudomonas aeruginosa methionyl-tRNA formyltransferase: importance of the base-base mismatch at the end of the acceptor stem

Formylation of the initiator methionyl-tRNA (Met-tRNAfMet) in eubacteria is catalyzed by methionyl-tRNA formyltransferase (MTF). Features of the Escherichia coli tRNAfMet that are important for formylation are the base-base mismatch between nucleotides 1 and 72, and the second and third base pairs of the acceptor stem. The base-base mismatch is the most crucial formylation determinant in the E. coli tRNAfMet. However, it is not known whether this feature is also important for formylation of other eubacterial tRNAfMet. We cloned the Pseudomonas aeruginosa MTF gene by complementation of an E. coli MTF mutant strain with a genomic library, and investigated the catalytic properties and substrate specificity of the enzyme. The results show that the P. aeruginosa and E. coli enzymes have comparable affinities for the tRNAfMet and N10-formyltetrahydrofolate (fTHF) substrates. Overproduction of the P. aeruginosa MTF rescued the initiator activity of an E. coli formylation-defective tRNAfMet with a base pair between nucleotides 1 and 72, indicating that the base-base mismatch is utilized by the P. aeruginosa MTF for recognition of the tRNAfMet. Therefore, this feature may be used by MTFs from other eubacteria to distinguish the initiator from elongator tRNAs.     

Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae.

The conserved positions of the eukaryotic cytoplasmic initiator tRNA have been suggested to be important for the initiation of protein synthesis. However, the role of these positions is not known. We describe in this report a functional analysis of the yeast initiator methionine tRNA (tRNA(iMet)), using a novel in vivo assay system which is not dependent on suppressor tRNAs. Strains of Saccharomyces cerevisiae with null alleles of the four initiator methionine tRNA (IMT) genes were constructed. Consequently, growth of these strains was dependent on tRNA(iMet) encoded from a plasmid-derived gene. We used these strains to investigate the significance of the conserved nucleosides of yeast tRNA(iMet) in vivo. Nucleotide substitutions corresponding to the nucleosides of the yeast elongator methionine tRNA (tRNA(MMet)) have been made at all conserved positions to identify the positions that are important for tRNA(iMet) to function in the initiation process. Surprisingly, nucleoside changes in base pairs 3-70, 12-23, 31-39, and 29-41, as well as expanding loop I by inserting an A at position 17 (A17) had no effect on the tester strain. Nucleotide substitutions in positions 54 and 60 to cytidines and guanosines (C54, G54, C60, and G60) did not prevent cell growth. In contrast, the double mutation U/rT54C60 blocked cell growth, and changing the A-U base pair 1-72 to a G-C base pair was deleterious to the cell, although these tRNAs were synthesized and accepted methionine in vitro. From our data, we suggest that an A-U base pair in position 1-72 is important for tRNA(iMet) function, that the hypothetical requirement for adenosines at positions 54 and 60 is invalid, and that a U/rT at position 54 is an antideterminant distinguishing an elongator from an initiator tRNA in the initiation of translation.     

The 51-63 base pair of tRNA confers specificity for binding by EF-Tu

Elongation factor Tu (EF-Tu) exhibits significant specificity for the different elongator tRNA bodies in order to offset its variable affinity to the esterified amino acid. Three X-ray cocrystal structures reveal that while most of the contacts with the protein involve the phosphodiester backbone of tRNA, a single hydrogen bond is observed between the Glu390 and the amino group of a guanine in the 51-63 base pair in the T-stem of tRNA. Here we show that the Glu390Ala mutation of Thermus thermophilus EF-Tu selectively destabilizes binding of those tRNAs containing a guanine at either position 51 or 63 and that mutagenesis of the 51-63 base pair in several tRNAs modulates their binding affinities to EF-Tu. A comparison of Escherichia coli tRNA sequences suggests that this specificity mechanism is conserved across the bacterial domain. While this contact is an important specificity determinant, it is clear that others remain to be identified.     

Interaction of methionine-specific tRNAs from Escherichia coli with immobilized elongation factor Tu

The interaction of three different Met-tRNAsMet from E. coli with bacterial elongation factor (EF) Tu X GTP was investigated by affinity chromatography. Met-tRNAfMet which lacks the base pair at the end of the acceptor stem binds only weakly to EF-Tu X GTP, while Met-tRNAmMet has a high affinity for the elongation factor. A modified Met-tRNAfMet which has a C1-G72 base pair binds much more strongly to immobilized EF-Tu X GTP than the native aminoacyl(aa)-tRNA with non-base-paired C1A72 at this position, demonstrating that the base pair including the first nucleotide in the tRNA is one of the essential structural requirements for the aa-tRNA X EF-Tu X GTP ternary complex formation.     

Structural features that underlie the use of bacterial Met-tRNAfMet primarily as an elongator in eukaryotic protein synthesis

Met-tRNAfMet from Escherichia coli is utilized efficiently as an elongator tRNA during protein synthesis in the rabbit reticulocyte lysate since it rapidly incorporates its methionyl residue into the same tryptic peptides of rabbit globin as the endogenous Met-tRNAmMet. Therefore, it must lack the structural characteristics that prevent the eukaryotic initiator tRNA from entering elongation. In contrast, E. coli Met-tRNAfMet appears to initiate very poorly since, unlike reticulocyte Met-tRNAiMet, it forms no detectable 43 S preinitiation complexes, and only a very small fraction of the methionine it contributes to polyribosomal peptidyl-tRNA is found at the N terminus. The bacterial fMet-tRNAfMet, which cannot elongate, is utilized for polypeptide chain initiation at a much lower level than the formylated Met-tRNAiMet from eukaryotes. The ability of E. coli Met-tRNAfMet to be used as an elongator and fMet-tRNAfMet as an initiator in the reticulocyte lysate may be considerably underestimated because of the rapid enzymatic hydrolysis of these initiator tRNAs in the lysate. The enzyme hydrolyzes fMet-tRNAfMet and Met-tRNAfMet from E. coli in a strictly Mg2+-dependent manner but not the corresponding species from yeast or rabbit reticulocytes. It also hydrolyzes yeast N-acetyl-Phe-tRNAPhe and reticulocyte peptidyl-tRNA, showing that this enzyme--like the eukaryotic protein synthetic machinery--does not readily distinguish the bacterial tRNAfMet from eukaryotic elongator tRNA.     

Decoding at the ribosomal A site: antibiotics, misreading and energy of aminoacyl-tRNA binding

The binding of Phe-tRNAPhe at the programmed ribosomal A site has been investigated using antibiotics that influence this binding in different ways. The adhesion of Phe-tRNAPhe, the consumption of GTP and the extent of the peptidyl transfer reaction were monitored. All of the five known misreading-inducing antibiotics that were tested stabilised the binding of Phe-tRNAPhe after its affixture to the A site by EF-Tu with GTP hydrolysis. The stabilisation was sufficient to overcome a single mismatch in the codon-anticodon interaction. Combinations of stabilising and destabilising influences were found to be additive, thus supporting the concepts: (1) that there is a 'correct' binding energy for aminoacyl tRNA in the A site, whose reduction hampers polypeptide synthesis and whose increase makes it inaccurate by by-passing proofreading; and (2) that the different antibiotics affect the bound aminoacyl tRNA at different points.     

Recognition of tRNAs by Methionyl-tRNA transformylase from mammalian mitochondria

Protein synthesis involves two methionine-isoaccepting tRNAs, an initiator and an elongator. In eubacteria, mitochondria, and chloroplasts, the addition of a formyl group gives its full functional identity to initiator Met-tRNA(Met). In Escherichia coli, it has been shown that the specific action of methionyl-tRNA transformylase on Met-tRNA(f)(Met) mainly involves a set of nucleotides in the acceptor stem, particularly a C(1)A(72) mismatch. In animal mitochondria, only one tRNA(Met) species has yet been described. It is admitted that this species can engage itself either in initiation or elongation of translation, depending on the presence or absence of a formyl group. In the present study, we searched for the identity elements of tRNA(Met) that govern its formylation by bovine mitochondrial transformylase. The main conclusion is that the mitochondrial formylase preferentially recognizes the methionyl moiety of its tRNA substrate. Moreover, the relatively small importance of the tRNA acceptor stem in the recognition process accounts for the protection against formylation of the mitochondrial tRNAs that share with tRNA(Met) an A(1)U(72) motif.     

Function of the extra 5'-phosphate carried by histidine tRNA

Among elongator tRNAs, tRNA specific for histidine has the peculiarity to possess one extra nucleotide at position -1. This nucleotide is believed to be responsible for recognition by histidyl-tRNA synthetase. Here, we show that, in fact, it is the phosphate 5' to the extra nucleotide which mainly supports the efficiency of the tRNA aminoacylation reaction catalyzed by Escherichia coli histidyl-tRNA synthetase. In the case of the reaction of E. coli peptidyl-tRNA hydrolase, this atypical phosphate is dispensable. Instead, peptidyl-tRNA hydrolase recognizes the phosphate of the phosphodiester bond between residues -1 and +1 of tRNA(His). Recognition of the +1 phosphate of tRNA(His) by peptidyl-tRNA hydrolase resembles, therefore, that of the 5'-terminal phosphate of other elongator tRNAs.     

Time-resolved fluorescence studies on the ternary complex formed between bacterial elongation factor Tu, guanosine 5'-triphosphate, and phenylalanyl-tRNAPhe

Time-resolved fluorescence spectroscopy was used to investigate the solution dynamics of Escherichia coli tRNAPhe, Phe-tRNAPhe, and Phe-tRNAPhe associated with GTP and elongation factor Tu (EF-Tu) in a ternary complex. Two fluorescence probes were employed: fluorescein, covalently bound to Phe-tRNAPhe at the s4U8 base (Phe-tRNAPhe-Fl8), and ethidium bromide, noncovalently associated with the tRNA (EB.Phe-tRNAPhe). The lifetimes observed for ethidium bromide were 1.89 ns, free in solution, and 26.3 ns, bound to its tight binding site on tRNA. Fluorescein-labeled tRNA had a lifetime of 4.3 ns, with no significant difference among the values for aminoacylated, unacylated, and EF-Tu-bound Phe-tRNAPhe-Fl8. Differential phase and modulation data for each fluorophore-tRNA system were fit with local and global Debye rotational relaxation times. Local motion of the labeled fluorescein in Phe-tRNAPhe-Fl8, tRNAPhe-Fl8, and Phe-tRNAPhe-Fl8.EF-Tu.GTP was characterized by rotational relaxation times of 2.7 +/- 0.5, 2.4 +/- 0.4, and 2.4 +/- 0.1 ns, respectively. These values are equal, within experimental error, and suggest that the rotational mobility of the s4U8-conjugated dye is unaffected by either tRNAPhe aminoacylation or ternary complex formation. Global rotational relaxation times for Phe-tRNAPhe-Fl8, 97 ns, and EB.Phe-tRNAPhe, 140 ns, were equivalent to those determined for the unacylated species, denoting little change in the overall size or shape of the tRNA molecule upon aminoacylation. These values for (Phe-)tRNA were larger than expected for a hydrated sphere of equivalent volume, 83 ns, and therefore confirm the asymmetric nature of the tRNA structure in solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and sequence elements important for recognition of Escherichia coli formylmethionine tRNA by methionyl-tRNA transformylase are clustered in the acceptor stem

We show that the structure and/or sequence of the first three base pairs at the end of the amino acid acceptor stem of Escherichia coli initiator tRNA and the discriminator base 73 are important for its formylation by E. coli methionyl-tRNA transformylase. This conclusion is based on mutagenesis of the E. coli initiator tRNA gene followed by measurement of kinetic parameters for formylation of the mutant tRNAs in vitro and function in protein synthesis in vivo. The first base pair found at the end of the amino acid acceptor stem in all other tRNAs is replaced by a C.A. "mismatch" in E. coli initiator tRNA. Mutation of this C.A. to U:A, a weak base pair, or U.G., a mismatch, has little effect on formylation, whereas mutation to C:G, a strong base pair, has a dramatic effect lowering Vmax/Kappm by 495-fold. Mutation of the second basepair G2:C71 to U2:A71 lowers Vmax/Kappm by 236-fold. Replacement of the third base-pair C3:G70 by U3:A70, A3:U70, or G3:C70 lowers Vmax/Kappm by about 67-, 27-, and 30-fold, respectively. Changes in the rest of the acceptor stem, dihydrouridine stem, anticodon stem, anticodon sequence, and T psi C stem have little or no effect on formylation.