Structure-function relationships in transfer RNA and methionine binding domains of Escherichia coli methionyl-tRNA synthetase

Abstract

Methionyl-tRNA synthetase (MetRS) shows a high specificity for its cognate substrates in the aminoacylation of methionine tRNAs. To determine the molecular basis for this specificity, I have used site-directed mutagenesis of a cloned MetRS gene to identify and study the role of amino acid residues comprising the tRNA and methionine binding domains of MetRS. Previous studies from this laboratory have shown that the anticodon of tRNA{dollar}\sp{lcub}\rm fMet{rcub}{dollar} is required for aminoacylation of tRNA by E. coli MetRS and have identified residue Trp461 in MetRS as a major element of the anticodon binding domain on the enzyme. In order to gain information about other possible amino acid residues involved in recognition of tRNA by MetRS, I have examined the effects of substitutions of other amino acid residues located on the surface of the carboxyl terminal domain of the enzyme. The data indicate that the amino acid residue Asn391 in MetRS is also involved in recognition of cognate tRNAs and interacts directly with the 3{dollar}\sp\prime{dollar} nucleotide of the anticodon of tRNA{dollar}\sp{lcub}\rm fMet{rcub}.{dollar} Molecular modeling and biochemical data suggest that recognition of tRNA{dollar}\sp{lcub}\rm fMet{rcub}{dollar} by MetRS involves a significant conformational change in the anticodon binding domain of the enzyme, which allows simultaneous contacts to be made between the anticodon loop and the two separate peptides containing Asn391-Arg395 and Trp461. In addition, second-site revertants of the mutant W461F MetRS that is able to aminoacylate an anticodon mutant of tRNA{dollar}\sp{lcub}\rm fMet{rcub}{dollar} were isolated using a genetic screen. One such revertant had a mutation at position 29 resulting in a change of Ile to Thr in addition to the original W461F mutation. This double mutant enzyme partially remedied the tRNA charging defect of the W461F mutant MetRS.;The roles of some amino acid residues located in the N-terminal domain and predicted to comprise the active site of MetRS have been examined in the binding of methionine, the specificity of the editing function and its relation to the synthetic function of MetRS in vitro. Hydrophobic interaction and hydrogen bonding by residues Trp305 and Tyr15, respectively, direct the cognate substrate methionine through the synthetic pathway and prevent it from entering the editing pathway. The W305A, Y15A, and Y15F mutant enzymes have lost the ability to discriminate against methionine in the editing reaction because the side chains of Trp305 and Tyr15 provide specific interactions with the side chain of methionine. The interactions of residues Arg233 and Asp52 of MetRS with the carboxyl and amino groups of methionine, respectively, which are essential for the synthetic function, are also essential for the editing function of the enzyme. These results also explain why the noncognate homocysteine is edited by MetRS.