Fluorine-19 nuclear magnetic resonance studies of the structure of 5-fluorouracil-substituted escherichia coli transfer RNA

¹⁹F nuclear magnetic resonance has been used to study fully active Escherichia coli tRNA₁^Val in which 5-fluorouracil has replaced more than 90% of all uracil and uracil-derived modified bases. The ¹⁹F spectrum of the native tRNA contains resolved resonances for all 14 incorporated 5-fluorouracils. These are spread over a 6 ppm range, from 1.8 to 7.7 ppm downfield of the standard free 5-fluorouracil. The ¹⁹F resonances serve as sensitive monitors of tRNA conformation. Removal of magnesium or addition of NaCl produces major, reversible changes in the ¹⁹F spectrum. Most affected is the lowest field resonance (peak A) in the spectrum of the native tRNA. This shifts 2–3 ppm upfield as the Mg²⁺ concentration is lowered or the NaCl concentration is raised. Thermal denaturation of the tRNA results in a collapse of the spectrum to a single broad peak centered at 4.7 ppm. Study of the pH dependence of the ¹⁹F spectrum shows that five incorporated fluorouracils with ¹⁹F signals in the central, 4-5.5 ppm, region of the spectrum, peaks C, D, E, F, and H, are accessible to titration in the pH 4,5-9 range. All have pK_a's close to that of free 5-fluorouridine (ca. 7.5). Evidence for a conformation change in the tRNA at mildly acidic pHs, ca. 5.5, is also presented. Four of the titratable 5-fluorouracil residues, those corresponding to peaks D, E/F, and H in the ¹⁹F spectrum of fluorine-labeled tRNA₁^Val, are essentially completely exposed to solvent as determined by the solvent isotope shift (SIS) on transfer of the tRNA from H₂0 to ²H₂0. These are also the 5-fluorouracils that readily form adducts with bisulfite, a reagent that reacts preferentially with pyrimidines in single-stranded regions. On the basis of these results, resonances D, E, F, and H in the middle of the ¹⁹F spectrum are attributed to 5-fluorouracils in non-base-paired (loop) regions of the tRNA. Evidence from the ionic strength dependence of the ¹⁹F spectrum and arguments based on other recent studies with fluorinated tRNAs support earlier suggestions [Horowitz, J., Ofengand, J., Daniel, W. E., & Cohn, M. (1977) J. Biol. Chem. 252, 4418–4420] that the resonances at lowest field correspond to tertiary hydrogen-bonded 5-fluorouracils. Consideration of ring-current effects and the preferential perturbation of upfield ¹⁹F resonances by the cyclophotoaddition of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen, which is known to react most readily with pyrimidines in double-stranded regions, permits initial assignment of upfield resonances to 5-fluorouracils in helical stems. The results demonstrate the promise of ¹⁹F nuclear magnetic resonance as a probe for the structure of tRNA in solution.