AFFINITY LABELING OF ESCHERICHIA COLI METHIONYL - TRANSFER-RNA SYNTHETASE

Abstract

The highly specific interaction of transfer RNA with aminoacyl-tRNA synthetases represents an attractive model system for studying the molecular basis of RNA-protein recognition. The 3D structure of tRNA('fMet) and a biologically active tryptic fragment of methionyl-tRNA synthetase have been solved and the sequences are known. Chemical crosslinking studies have been carried out in this laboratory in order to determine the orientations of the two macromolecules within the protein-nucleic acid complex.;Previous studies identified four crosslinked peptides at the tRNA binding site of MetRS. In the present work, the sites in the tRNA to which the identified peptides are crosslinked have been determined. The results indicate that peptides containing Lys 402 and Lys 439 are crosslinked to the D-loop, Lys 465 is crosslinked to the anticodon and Lys 640 to the 5' end of the tRNA.;A lysine-reactive crosslinker has been coupled to the amino group of the rare base 3-(3-amino-3-carboxypropyl) uridine (X) in tRNA(,m)('Met). Affinity labeling studies of MetRS with this derivative showed a stoichiometric crosslinking. The coupling reaction is specific and one major peptide has been isolated and sequenced, corresponding to Lys 596.;A new bifunctional crosslinker has been synthesized. This reagent can be specifically coupled to the 4-thiouridine (s('4)U) in tRNA('fMet). Reaction of the derivatized tRNA with MetRS results in the stoichiometric and specific coupling of tRNA to lysine residues in the protein. This method can be extended to other synthetases which bind tRNAs having the s('4)U base.;The crosslinking data, in combination with the refined crystal structure of the enzyme (1.8 (ANGSTROM)) will allow the construction of the first detailed model of a tRNA-synthetase complex.