RECOGNITION OF ESCHERICHIA-COLI VALINE TRANSFER-RNA BY ITS COGNATE SYNTHETASE - A F-19 NMR-STUDY

Interactions of 5-fluorouracil-substituted Escherichia coli tRNA(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 [Heck, J.D., & Hatfield, G.W. (1988) J. Biol. Chem. 263, 868-877]. Apparent K(M) and V(max) values of the purified VRS are the same for both normal and 5-fluorouracil (FUra)-substituted tRNA(Val). Binding of VRS to (FUra)tRNA(Val) induces structural perturbations that are reflected in selective changes in the F-19 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(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(Val), suggesting conformational changes in this part of the molecule. No F-19 NMR evidence was found for formation of the Michael adduct between VRS and FU8 of 5-fluorouracil-substituted tRNA(Val) that has been proposed as a common intermediate in the aminoacylation reaction.