Partial Assignment of Resonances in the ¹⁹F Nuclear Magnetic Resonance Spectra of 5-Fluorouracil-Substituted Transfer RNAs

Features of the¹⁹F nuclear magnetic resonance (NMR) spectra of three purified 5-fluorouracil-(FUra-) substituted Escherichia coli tRNAs, tRNA₁^val, tRNA_m^Met, and tRNA_f^Met, are compared. Each of the tRNA species can be resolved into two isoaccepting forms, A and B, whose¹⁹F NMR spectra differ in the shift of one peak from the 4.5 to 4.8 parts per million (ppm) range (FUra = 0) in the spectrum of isoacceptor B upfield to ca. -15 ppm in that of isoacceptor A. Because the sequences of the two isoacceptors of each tRNA differ only at one position in the D loop, that normally occupied by a dihydrouridine residue, we assign the 4.5 ppm peak in the spectrum of fluorine-labeled tRNA₁^Valto FUra 17 and the resonance at 4.6 ppm in the spectrum of fluorouracil-substituted tRNA_m^metto FUra20. A reciprocal¹⁹F{^l9F} nuclear Overhauser effect is observed between the downfield peaks A and B in the^l9F NMR spectrum of¹⁹F-labeled tRNA₁^val. Assuming that fluorine-labeled tRNA₁^valhas a structure similar to that of yeast tRNA^Phe, only FUra54 and -55 are close enough (4–5 A) to give an appreciable¹⁹F homonuclear Overhauser effect. Peaks A and B have therefore been assigned to FUra54 and -55. As the temperature is raised from 30 to 45 °C, the intensity of peak B (6.6 ppm) in the spectrum of¹⁹F-labeled tRNA₁^valgradually shifts upfield to 6.4 ppm (T_m= 36 °C), indicating a temperature-dependent slow exchange of the corresponding 5-fluorouracil residue between two magnetically distinct environments. Because this effect resembles the splitting of T54 methyl¹H and¹³C signals in the spectra of several native tRNAs [Kastrup, R. V., & Schmidt, P. G. (1978) Nucleic Acids Res. 5, 257-269; Kopper, R. A., Schmidt, P. G., & Agris, P. F. (1983) Biochemistry 22, 1396–1401], we assign peak B to FUra54. Peak A in the spectrum of¹⁹F-labeled tRNA₁^valcan then be assigned to FUra55. The lowest field resonance (peak A) in the¹⁹F spectra of each of the three tRNAs exhibits a uniquely large chemical shift change with changing ionic strength or magnesium ion concentration. This similarity suggests that peak A corresponds to a conserved base in the tRNAs and is consistent with assignment of peak A in the¹⁹F NMR spectra of all three fluorinated tRNAs to the 5-fluorouracil residue that replaces the invariant Ψ55.