Modification of specific lysine residues in E. coli methionyl-tRNA synthetase by crosslinking to E. coli formylmethionine tRNA.
A protein affinity labeling derivative of E. coli tRNAfMet has been prepared which carries an average of one reactive side chain per molecule, distributed over four structural regions. Each side chain contains a disulfide bond capable of reaction with cysteine residues and an N-hydroxysuccinimide ester group capable of coupling to lysine epsilon-amino groups in proteins. Reaction of the modified tRNA with E. coli methionyl-tRNA synthetase leads to crosslinking only by reaction with lysine residues in the protein. Examination of the tRNA present in the crosslinked complex reveals that the enzyme is coupled to side chains attached to the 5' terminal nucleotide, the dihydrouridine loop, the anticodon and the CCA sequence. Digestion of the crosslinked enzyme with trypsin followed by peptide mapping reveals that the major crosslinking reactions occur at four specific lysine residues, with minor reaction at two additional sites. Native methionyl-tRNA synthetase contains 90 lysine residues, 45 in unique sequences of the dimeric alpha 2 enzyme. Crosslinking of the protein to different regions in tRNAfMet thus occurs with the high degree of selectivity necessary for use in determining the peptide sequences which are near specific nucleotide sequences of tRNA bound to the protein.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008239:Lysine; D008718:Methionine-tRNA Ligase / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012358:RNA, Transfer, Met; D013388:Succinimides / Q000494:pharmacology*
D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; D013388:Succinimides; C024988:tRNA, formylmethionine-; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase; C011240:dithiobis(succinimidylpropionate); D008239:Lysine