Identification of the tRNA anticodon recognition site of Escherichia coli methionyl-tRNA synthetase.
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)
Ghosh
G
G
Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, Bronx, New York 10461.
Pelka
H
H
Schulman
L H
LH
D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics*; D000926:Anticodon / Q000378:metabolism; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D008718:Methionine-tRNA Ligase / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D012343:RNA, Transfer / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D000926:Anticodon; D012346:RNA, Transfer, Amino Acyl; C026464:tRNA(m)(Met), methionine-; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase