Recognition of Escherichia coli valine transfer RNA by its cognate synthetase: A fluorine-19 NMR study

Journal Article · Tue Feb 12 04:00:00 EST 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00220a031· OSTI ID:5510355

Chu, Wenchy; Horowitz, J ^[1]

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1. Iowa State Univ., Ames (USA)

Interactions of 5-fluorouracil-substituted Escherichia coli tRNA{sup Val} with its cognate synthetase have been investigated by fluorine-19 nuclear magnetic resonance. Valyl-tRNA synthetase (VRS) (EC 6.1.1.9), purified to homogeneity from an overproducing strain of E. coli, differs somewhat from VRS previously isolated from E. coli K12. Its amino acid composition and N-terminal sequence agree well with results derived from the sequence of the VRS gene. Apparent K{sub M} and V{sub max} values of the purified VRS are the same for both normal and 5-fluorouracil (FUra)-substituted tRNA{sup Val}. Binding of VRS to (FUra)tRNA{sup Val} induces structural perturbations that are reflected in selective changes in the {sup 19}F NMR spectrum of the tRNA. Addition of increasing amounts of VRS results in a gradual loss of intensity at resonances corresponding to FU34, FU7, and FU67, with FU34, at the wobble position of the anticodon, being affected most. At higher VRS/tRNA ratios, a broadening and shifting of FU12 and of FU4 and/or FU8 occur. These results indicate that VRS interacts with tRNA{sup Val} along the entire inside of the L-shape molecule, from the acceptor stem to the anticodon. Valyl-tRNA synthetase also causes a splitting of resonances FU55 and FU64 in the T-loop and stem of tRNA{sup Val}, suggesting conformational changes in this part of the molecule. No {sup 19}F NMR evidence was found for formation of the Michael adduct between VRS and FU8 of 5-fluorouracil-substituted tRNA{sup Val} that has been proposed as a common intermediate in the aminoacylation reaction.

• OSTI ID: 5510355
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 30:6; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ADDUCTS
AMINO ACIDS
BACTERIA
CARBOXYLIC ACIDS
DNA ADDUCTS
ELECTROPHORESIS
ENZYMES
ESCHERICHIA COLI
FLUORINE 19
FLUORINE ISOTOPES
ISOTOPES
LIGASES
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
NMR SPECTRA
NUCLEAR MAGNETIC RESONANCE
NUCLEI
NUCLEIC ACIDS
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
PURIFICATION
RESONANCE
RNA
SPECTRA
STABLE ISOTOPES
TRANSFER RNA
VALINE