Affinity labeling of Escherichia coli phenylalanyl-tRNA synthetase at the binding site for tRNA
Periodate-oxidized tRNA/sup Phe/ (tRNA/sub ox//sup Phe/) behaves as a specific affinity label of tetrameric Escherichia coli phenylalanyl-tRNA synthetase (PheRS). Reaction of the ..cap alpha../sub 2/..beta../sub 2/ enzyme with tRNA/sub ox//sup Phe/ results in the loss of tRNA/sup Phe/ 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-(/sup 14/C)tRNA/sub ox//sup Phe/ covalent complex indicates that the large (..cap alpha.., M/sub r/ 87K) subunit of the enzyme interacts with the 3'-adenosine of tRNA/sub ox//sup Phe/. The (/sup 14/C)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 ..cap 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.
- Research Organization:
- Ecole Polytechnique, Palaiseau, France
- OSTI ID:
- 5998344
- Journal Information:
- Biochemistry; (United States), Vol. 26:17
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
62 RADIOLOGY AND NUCLEAR MEDICINE
LIGASES
CONFIGURATION INTERACTION
PEPTIDES
AMINO ACID SEQUENCE
AUTORADIOGRAPHY
TRANSFER RNA
CARBON 14 COMPOUNDS
ELECTROPHORESIS
ESCHERICHIA COLI
PHENYLALANINE
PURIFICATION
AMINO ACIDS
BACTERIA
CARBOXYLIC ACIDS
ENZYMES
LABELLED COMPOUNDS
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
PROTEINS
RNA
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