Recognition of Escherichia coli valine transfer RNA by its cognate synthetase: a fluorine-19 NMR study.
Interactions of 5-fluorouracil-substituted Escherichia coli tRNAVal 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 KM and Vmax values of the purified VRS are the same for both normal and 5-fluorouracil (FUra)-substituted tRNAVal. Binding of VRS to (FUra)tRNAVal induces structural perturbations that are reflected in selective changes in the 19F 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 tRNAVal 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 tRNAVal, suggesting conformational changes in this part of the molecule. No 19F NMR evidence was found for formation of the Michael adduct between VRS and FU8 of 5-fluorouracil-substituted tRNAVal that has been proposed as a common intermediate in the aminoacylation reaction.
Chu
W C
WC
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Horowitz
J
J
D001483:Base Sequence; D001665:Binding Sites; D002845:Chromatography; D002850:Chromatography, Gel; D017886:Durapatite; D004926:Escherichia coli / Q000378:metabolism*; D005461:Fluorine; D005472:Fluorouracil; D006882:Hydroxyapatites; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012366:RNA, Transfer, Val / Q000378:metabolism*; D014637:Valine-tRNA Ligase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D006882:Hydroxyapatites; D012366:RNA, Transfer, Val; D005461:Fluorine; D017886:Durapatite; D014637:Valine-tRNA Ligase; D005472:Fluorouracil