Substrate recognition by Escherichia coli methionyl-tRNA synthetase
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Abstract
Methionyl-tRNA synthetase exhibits a high specificity for its cognate substrates in the aminoacylation tRNA{dollar}\sp{lcub}\rm Met{rcub}{dollar}. High resolution crystal structures of the biologically active truncated methionyl-tRNA synthetase (MetRS) and of the tRNA{dollar}\sp{lcub}\rm fMet{rcub}{dollar} are available.;Conversion of Trp461, located in the anticodon recognition domain of MetRS, to Phe had a significant effect on aminoacylation. The mutant enzyme showed a 60-100 fold increase in Km for methionine tRNAs with no change in k{dollar}\sb{lcub}\rm cat{rcub}{dollar}. Conversion of the adjacent Pro460 to Leu resulted in a small but significant increase in the Km for tRNA. The kinetic parameters for methionine and ATP were unaltered in both cases. Examination of the aminoacylation of anticodon derivatives of tRNA{dollar}\sp{lcub}\rm Met{rcub}{dollar} by the mutant enzymes revealed sequence specific interactions between the Phe461 mutant and different anticodons which suggest that Trp461 may directly interact with the anticodon base C34. An Asp456 to Asn change altered the Km for tRNA{dollar}\sp{lcub}\rm fMet{rcub}{dollar} but not tRNA{dollar}\sp{lcub}\rm Met{rcub}\sb{lcub}\rm m{rcub}{dollar}.;Several positively charged surface residues located in regions of MetRS which are structurally homologous to domains of GlnRS were mutated to examine their role in the aminocylation reaction.;Trp305 and Arg233 affect the initial binding of methionine, however Arg233 is also involved in the catalysis of methionyl adenylate formation. The data suggest that Asp52 may interact with methionine in the transition state leading to aminoacyl adenylate formation. Mutation of Tyr359 alters the Km for ATP in the activation reaction whereas the Km's for methionine and tRNA remain unaltered. An adjacent Tyr residue does not affect the Km for substrate but dramatically lowers k{dollar}\sb{lcub}\rm cat{rcub}{dollar} for aminoacyl adenylate formation.;E. coli cells carrying a plasmid containing tRNA{dollar}\sp{lcub}\rm fMet{rcub}{dollar} with a GGU anticodon in tandem with a lac{dollar}\sp{lcub}\rm Z{rcub}{dollar} gene having the complementary ACC initiation codon fail to produce {dollar}\beta{dollar}-galactosidase unless supplemented with a mutant MetRS that can aminoacylate the mutant initiator tRNA. One mutant MetRS gene was isolated three times using this screen. The nucleotide sequence showed a cluster of mutations within a six nucleotide window leading to a +1 frameshift at amino acid position 194. Western blot analysis indicated that low levels of full length MetRS547 are produced from the mutant. (Abstract shortened with permission of author.).