Covalent coupling of the variable loop of the elongator methionine tRNA to a specific lysine residue in Escherichia coli methionyl-tRNA synthetase.
A lysine-reactive cross-linker has been coupled to the minor base 3-(3-amino-3-carboxypropyl)uridine in the variable loop of the Escherichia coli elongator methionine tRNA (tRNA(mMet]. Incubation of the derivatized tRNA with E. coli methionyl-tRNA synthetase (MetRS) resulted in covalent coupling of the protein and nucleic acid and loss of amino acid acceptor activity of the enzyme. One mole of tRNA was cross-linked per mole of enzyme inactivated. Enzyme activity was largely restored by release of the bound tRNA following cleavage of the disulfide bond in the cross-linker with a sulfhydryl reagent. The cross-linking reaction was effectively inhibited by unmodified tRNA(mMet) but not by noncognate tRNA(Phe). The covalent complex was digested with trypsin, and the resulting tRNA-bound peptides were isolated by anion-exchange chromatography. The cross-linked peptides were released from the tRNA by cleavage in the disulfide bond of the cross-linker and purified by reverse-phase high-pressure liquid chromatography, yielding one major peptide plus several minor peptides. Amino acid analysis indicated that the major product was an octadecapeptide cross-linked to tRNA(mMet) through lysine residue 596 in the primary sequence of MetRS. The N-terminal sequence of the peptide was determined to be Val-Ala-Leu-Ile-Glu-Asn-Ala-Glu-Phe-Val, corresponding to residues 582-591 in MetRS. The procedures described here should be applicable to the determination of peptide sequences near the variable loop of other tRNAs containing the 3-(3-amino-3-carboxypropyl)uracil base when such tRNAs are bound to specific proteins.
D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008239:Lysine; D008718:Methionine-tRNA Ligase / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*
D012346:RNA, Transfer, Amino Acyl; C026464:tRNA(m)(Met), methionine-; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase; D008239:Lysine