Two separate peptides in Escherichia coli methionyl-tRNA synthetase form the anticodon binding site for methionine tRNA.
The amino acid residues Asn391, Arg395, and Trp461 in methionyl-tRNA synthetase (MetRS) of Escherichia coli are involved in the anticodon-dependent recognition of its cognate tRNAs [Ghosh, G., Pelka, H., & Schulman, L.H. (1990) Biochemistry 29, 2220-2225; Ghosh, G., Kim, H.Y., Demaret, J. P., Brunie, S., & Schulman, L.H. (1991) Biochemistry 30, 11767-11774]. While tryptophan at position 461 was shown to bind directly to the wobble base at position 34 in the tRNA(Met) anticodon, the role of residues 391-395 was not thoroughly explored. To gain further insight into the role of the 391-395 residues and nearby residues, appropriate mutations were analyzed for aminoacylation activity, as well as tRNA binding. Mutations of the phylogenetically conserved asparagine at position 391 increased the Km for aminoacylation of tRNA(Met) 18-40-fold. Further analysis using fluorescence titration indicated that the mutation affected initial complex formation, since the Kd for tRNA(Met) binding had increased at least 15-fold over wild type. Kinetic analysis of mutationally altered derivatives of MetRS with a series of tRNA(Met) derivatives containing base substitutions in the anticodon revealed sequence-specific interaction between the amino acid residue at position 391 and the U36 of the anticodon of tRNA(Met). In addition to position 391, position 387 was also found to affect tRNA(Met) binding and aminoacylation, indicating a possible significant role in interaction of the enzyme with the anticodon of tRNA(Met). These results indicate that the peptide segment containing residues 391-395 is involved in the direct recognition of the 3' end of the anticodon.
Kim
H Y
HY
Department of Developmental Biology & Cancer, Albert Einstein College of Medicine, Bronx, New York 10461.
Pelka
H
H
Brunie
S
S
Schulman
L H
LH
D000215:Acylation; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000926:Anticodon; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008718:Methionine-tRNA Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000378:metabolism*
D000926:Anticodon; D010446:Peptide Fragments; D010710:Phosphates; D012358:RNA, Transfer, Met; D000255:Adenosine Triphosphate; D008718:Methionine-tRNA Ligase