tRNA recognition site of Escherichia coli methionyl-tRNA synthetase.
We have previously shown that anticodon bases are essential for specific recognition of tRNA substrates by Escherichia coli methionyl-tRNA synthetase (MetRS) [Schulman, L. H., & Pelka, H. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6755-6759] and that the enzyme tightly binds to C34 at the wobble position of E. coli initiator methionine tRNA (tRNAfMet) [Pelka, H., & Schulman, L. H. (1986) Biochemistry 25, 4450-4456]. We have also previously demonstrated that an affinity labeling derivative of tRNAfMet can be quantitatively cross-linked to the tRNA binding site of MetRS [Valenzuela, D., & Schulman, L. H. (1986) Biochemistry 25, 4555-4561]. Here, we have determined the site in MetRS which is cross-linked to the anticodon of tRNAfMet, as well as the location of four additional cross-links. Only a single peptide, containing Lys465, is covalently coupled to C34, indicating that the recognition site for the anticodon is close to this sequence in the three-dimensional structure of MetRS. The D loop at one corner of the tRNA molecule is cross-linked to three peptides, containing Lys402, Lys439, and Lys596. The 5' terminus of the tRNA is cross-linked to Lys640, near the carboxy terminus of the enzyme. Since the 3' end of tRNAfMet is positioned close to the active site in the N-terminal domain [Hountondji, C., Blanquet, S., & Lederer, F. (1985) Biochemistry 24, 1175-1180], this result indicates that the carboxy ends of the two polypeptide chains of native dimeric MetRS are folded back toward the N-terminal domain of each subunit.
D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D008718:Methionine-tRNA Ligase / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011487:Protein Conformation; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012358:RNA, Transfer, Met; D006163:Ribonuclease T1; D014357:Trypsin
D010446:Peptide Fragments; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D006163:Ribonuclease T1; D014357:Trypsin; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase