Binding of the yeast tRNA(Met) anticodon by the cognate methionyl-tRNA synthetase involves at least two independent peptide regions.
As for Escherichia coli methionine tRNAs, the anticodon triplet of yeast tRNA(Met) plays an important role in the recognition by the yeast methionyl-tRNA synthetase (MetRS), indicating that this determinant for methionine identity is conserved in yeast. Efficient aminoacylation of the E. coli tRNA(Met) transcript by the heterologous yeast methionine enzyme also suggests conservation of the protein determinants that interact with the CAU anticodon sequence. We have analysed by site-directed mutagenesis the peptide region 655 to 663 of the yeast MetRS that is equivalent to the anticodon binding region of the E. coli methionine enzyme. Only one change, converting Leu658 into Ala significantly reduced tRNA aminoacylation. Semi-conservative substitutions of L658 allow a correlation to be drawn between side-chain volume of the hydrophobic residue at this site and activity. The analysis of the L658A mutant shows that Km is mainly affected. This suggests that the peptide region 655 to 663 contributes partially to the binding of the anticodon, since separate mutational analysis of the anticodon bases shows that kcat is the most critical parameter in the recognition of tRNA(Met) by the yeast synthetase. We have analysed the role of peptide region (583-GNLVNR-588) that is spatially close to the region 655 to 663. Replacements of residues N584 and R588 reduces significantly the kcat of aminoacylation. The peptide region 583-GNLVNR-588 is highly conserved in all MetRS so far sequenced. We therefore propose that the hydrogen donor/acceptor amino acid residues within this region are the most critical protein determinants for the positive selection of the methionine tRNAs.
Despons
L
L
Unité Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance IBMC du CRNS, Strasbourg, France.
Senger
B
B
Fasiolo
F
F
Walter
P
P
D000595:Amino Acid Sequence; D000926:Anticodon / Q000378:metabolism*; D001665:Binding Sites; D007700:Kinetics; D008718:Methionine-tRNA Ligase / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012358:RNA, Transfer, Met / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013329:Structure-Activity Relationship
D000926:Anticodon; D012358:RNA, Transfer, Met; D008718:Methionine-tRNA Ligase