Fluorine-19 NMR studies of the thermal unfolding of 5-fluorouracil-substituted Escherichia coli valine transfer RNA

Abstract

19F NMR spectroscopy was used to monitor the thermal unfolding of E. coli tRNA Val labeled by incorporation of 5-fluorouracil (FUra). With rising temperatures, resonances in the 19F NMR spectrum of (FUra)tRNA Val gradually shift towards the central region of the spectrum and merge into a single broad peak above 85°C. FU55 and FU12 are the first to shift, beginning at temperatures below 40°C, which suggests that the initial steps of thermal denaturation of tRNA Val involve disruption of the tertiary interactions between the D- and T-arms. The acceptor stem and the FU64-G50 wobble base pair in the T-stem are particularly stable to thermal denaturation. A temperature-dependent splitting of the 19F resonance assigned to FU64, at temperatures above 40°C, suggests that the T-arm of (FUra)tRNA Val exists in two conformations in slow exchange on the NMR time scale.