Affinity labeling of Escherichia coli phenylalanyl-tRNA synthetase at the binding site for tRNAPhe

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Abstract
Periodate-oxidized tRNAPhe (tRNAoxPhe) behaves as a specific affinity label of tetrameric Escherichia coli phenylalanyl-tRNA synthetase (PheRS). Reactin of the .alpha.2.beta.2 enzyme with tRNAoxPhe results in the loss of tRNAPhe aminoacylation activity with covalent attachment of 2 mol of tRNA dialdehyde/mol of enzyme, in agreement with the stoichiometry of tRNA binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PheRS-[14C]tRNAoxPhe. The [14C]tRNA-labeled chymotryptic peptides of PheRS were purified by both gel filtration and reverse-phase high-performance liquid chromatography. The radioactivity was almost equally distributed among three peptides: Met-Lys[Ado]-Phe, Ala-Asp-Lys[Ado]-Leu, and Lys-Lle-Lys[Ado]-Ala. These sequences correspond to residues 1-3, 59-62, and 104-107, respectively, in the N-terminal region of the 795 amino acid sequence of the .alpha. subunit. It is noticeable that the labeled peptide Ala-Asp-Lys-Leu is adjacent to residues 63-66 (Arg-Val-Thr-Lys). The latter sequence was just predicted to resemble the proposed consensus tRNA CCA binding region Lys-Met-Ser-Lys-Ser, as deduced from previous affinity labeling studies on E. coli methionyl- and tyrosyl-tRNA synthases [Hountondji, C., Dessen, P., and Blanquet, S. (1986) Biochimie 68, 1071-1078].