Covalent coupling of 4-thiouridine in the initiator methionine tRNA to specific lysine residues in Escherichia coli methionyl-tRNA synthetase.
A new method has been developed to couple a lysine-reactive cross-linker to the 4-thiouridine residue at position 8 in the primary structure of the Escherichia coli initiator methionine tRNA (tRNAfMet). Incubation of the affinity-labeling tRNAfMet derivative with E. coli methionyl-tRNA synthetase (MetRS) yielded a covalent complex of the protein and nucleic acid and resulted in loss of amino acid acceptor activity of the enzyme. A stoichiometric relationship (1:1) was observed between the amount of cross-linked tRNA and the amount of enzyme inactivated. Cross-linking was effectively inhibited by unmodified tRNAfMet, but not by noncognate tRNAPhe. The covalent complex was digested with trypsin, and the resulting tRNA-bound peptides were purified from excess free peptides by anion-exchange chromatography. The tRNA was then degraded with T1 ribonuclease, and the peptides bound to the 4-thiouridine-containing dinucleotide were purified by high-pressure liquid chromatography. Two major peptide products were isolated plus several minor peptides. N-Terminal sequencing of the peptides obtained in highest yield revealed that the 4-thiouridine was cross-linked to lysine residues 402 and 439 in the primary sequence of MetRS. Since many prokaryotic tRNAs contain 4-thiouridine, the procedures described here should prove useful for identification of peptide sequences near this modified base when a variety of tRNAs are bound to specific proteins.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D062365:Aminobenzoates / Q000138:chemical synthesis* / Q000378:metabolism; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000138:chemical synthesis*; D004926:Escherichia coli / Q000201:enzymology*; D007202:Indicators and Reagents; D008239:Lysine; D008718:Methionine-tRNA Ligase / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012358:RNA, Transfer, Met; D013388:Succinimides / Q000138:chemical synthesis* / Q000378:metabolism; D013891:Thiouridine; D062366:para-Aminobenzoates
D062365:Aminobenzoates; D003432:Cross-Linking Reagents; D007202:Indicators and Reagents; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; D013388:Succinimides; D062366:para-Aminobenzoates; C024988:tRNA, formylmethionine-; D013891:Thiouridine; C054840:N-succinimidyl ((bromoacetyl)amino)benzoate; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase; D008239:Lysine