The anticodon has previously been shown to play a role in recognition of certain transfer RNAs by aminoacyl-tRNA synthetases; however, the extent to which this sequence dictates tRNA identity is generally unknown. To investigate the contribution of the anticodon to the identity of Escherichia coli methionine and valine tRNAs, in vitro transcripts of these tRNAs were prepared that contained normal and interchanged anticodon sequences. Transcripts containing wild-type tRNA sequences were excellent substrates for their respective cognate aminoacyl-tRNA synthetases and were effectively discriminated against by a variety of noncognate enzymes. The mutant tRNAs produced by switching the anticodon sequences lost their original tRNA identity and assumed an identity corresponding to the acquired anticodon sequence. These results indicate that the anticodon contains sufficient information to distinguish methionine and valine tRNAs with high fidelity.

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We have previously shown that the anticodon of methionine tRNAs contains most, if not all, of the nucleotides required for specific recognition of tRNA substrates by Escherichia coli methionyl-tRNA synthetase [Schulman, L. H., & Pelka, H. (1988) Science 242, 765-768]. Previous cross-linking experiments have also identified a site in the synthetase that lies within 14 A of the anticodon binding domain [Leon, O., & Schulman, L. H. (1987) Biochemistry 26, 5416-5422]. In the present work, we have carried out site-directed mutagenesis of this domain, creating conservative amino acid changes at residues that contain side chains having potential hydrogen-bond donors or acceptors. Only one of these changes, converting Trp461----Phe, had a significant effect on aminoacylation. The mutant enzyme showed an approximately 60-100-fold increase in Km for methionine tRNAs, with little or no change in the Km for methionine or ATP or in the maximal velocity of the aminoacylation reaction. Conversion of the adjacent Pro460 to Leu resulted in a smaller increase in Km for tRNA(Mets), with no change in the other kinetic parameters. Examination of the interaction of the mutant enzymes with a series of tRNA(Met) derivatives containing base substitutions in the anticodon revealed sequence-specific interactions between the Phe461 mutant and different anticodons. Km values were highest for tRNA(mMet) derivatives containing the normal anticodon wobble base C. Base substitutions at this site decreased the Km for aminoacylation by the Phe461 mutant, while increasing the Km for the wild-type enzyme and for the Leu460 mutant to values greater than 100 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

The contribution of the anticodon to the discrimination between cognate and noncognate tRNAs by Escherichia coli Arg-tRNA synthetase has been investigated by in vitro synthesis and aminoacylation of elongator methionine tRNA (tRNA(mMet) mutants. Substitution of the Arg anticodon CCG for the Met anticodon CAU leads to a dramatic increase in Arg acceptance by tRNA(mMet). A nucleotide (A20) previously identified by others in the dihydrouridine loop of tRNA(Arg)s makes a smaller contribution to the conversion of tRNA(mMet) identity from Met to Arg. The combined anticodon and dihydrouridine loop mutations yield a tRNA(mMet) derivative that is aminoacylated with near-normal kinetics by the Arg-tRNA synthetase.

We have analyzed, by site-directed mutagenesis, the molecular basis of the editing function and its relation to the synthetic function of Escherichia coli methionyl-tRNA synthetase. The data obtained fit a model of the active site that partitions an amino acid substrate between synthetic and editing pathways. Hydrophobic and hydrogen bonding interactions direct the cognate substrate methionine through the synthetic pathway and prevent it from entering the editing pathway. Two hydrophobic interactions are proposed: between the side chain of Trp-305 and a methyl group ofmethionine and between the benzene ring of Tyr-15 and the 1& and f-CH2 groups of the substrate. An essential hydrogen bond forms between the OH of Tyr-15 and an electron pair of the sulfur atom of methionine. Consistent with these functions, side chains of Trp-305 and Tyr-15 are localized on opposite sides of the cavity forming a putative methionine binding pocket that is observed in the three-dimensional crystallographic structure of methionyl-tRNA synthetase. Enzymes W305A, Y15A, and Y15F have diminished ability to discriminate against homocysteine in the synthetic reaction, compared to the wild-type enzyme. At the same time, mutant enzymes have lost the ability to discriminate against methionine in the editing reaction and edited Met-AMP to a similar extent as Hcy-AMP. Interactions of residues Arg-233 and Asp-52 of methionyl-tRNA synthetase with the carboxyl and amino groups, respectively, of the substrate, which are essential for the synthetic function, were also essential for the editing function of the enzyme. Deacylation of Met-tRNA to S-methylhomocysteine thiolactone catalyzed by W305A, Y15A, and Y15F mutant enzymes was only slightly impaired relative to the wild-type enzyme. However, enzymes R233Q, R233A, and D52A did not deacylate MettRNA. The model also explains why the noncognate homocysteine is edited by methionyl-tRNA synthetase.