Affinity labeling of Escherichia coli phenylalanyl-tRNA synthetase at the binding site for tRNAPhe.
Periodate-oxidized tRNA(Phe) (tRNA(oxPhe)) behaves as a specific affinity label of tetrameric Escherichia coli phenylalanyl-tRNA synthetase (PheRS). Reaction of the alpha 2 beta 2 enzyme with tRNA(oxPhe) 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]tRNA(oxPhe) covalent complex indicates that the large (alpha, Mr 87K) subunit of the enzyme interacts with the 3'-adenosine of tRNA(oxPhe). 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-Ile-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 synthetases [Hountondji, C., Dessen, P., & Blanquet, S. (1986) Biochimie 68, 1071-1078].
Hountondji
C
C
Laboratoire de Biochimie, Ecole Polytechnique, Palaiseau, France.
Schmitter
J M
JM
Beauvallet
C
C
Blanquet
S
S
D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis; D010504:Periodic Acid / Q000378:metabolism; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism*; D011485:Protein Binding; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*
D000345:Affinity Labels; D046911:Macromolecular Substances; D010446:Peptide Fragments; D012346:RNA, Transfer, Amino Acyl; D010504:Periodic Acid; D000604:Amino Acyl-tRNA Synthetases; D010652:Phenylalanine-tRNA Ligase