Reversible inactivation of Escherichia coli methionyl-tRNA synthetase by covalent attachment of formylmethionine tRNA to the tRNA binding site with a cleavable cross-linker.
Protein affinity labeling groups have been attached to single-stranded cytidine residues in four structural regions of tRNAfMet. Modification of the tRNA with an average of one cross-linking group per molecule is achieved with retention of 75% of the original methionine acceptor activity. Incubation of the modified tRNA with methionyl-tRNA synthetase (MetRS) results in covalent coupling of the protein and nucleic acid by reaction of N-hydroxysuccinimide ester groups attached to the tRNA with lysine residues in the enzyme. In the presence of excess MetRS, approximately 30% of the input tRNA can be covalently bound to protein, indicating that lysine residues are appropriately oriented for reaction with cross-linking groups attached to certain sites in the tRNA but not to others. The cross-linking reaction results in loss of aminoacylation activity of MetRS equal to the amount of covalently bound tRNA. Enzyme activity is restored by release of bound tRNA following cleavage of the disulfide bond of the cross-linker with a sulfhydryl reagent. The data indicate that cross-linking occurs at the tRNA binding site of the enzyme. In the presence of excess modified tRNAfMet, a maximum of 1 mol of tRNA is cross-linked per mol of MetRS, in keeping with the known anticooperative tRNA binding properties of the native dimeric synthetase. In addition, the coupling reaction is effectively inhibited by unmodified tRNAfMet, but not by noncognate tRNAs.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008718:Methionine-tRNA Ligase / Q000378:metabolism*; D011485:Protein Binding; D012343:RNA, Transfer / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000494:pharmacology*; D012358:RNA, Transfer, Met; D013388:Succinimides / Q000494:pharmacology
D003432:Cross-Linking Reagents; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; D013388:Succinimides; C024988:tRNA, formylmethionine-; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase; C011240:dithiobis(succinimidylpropionate)