Partial assignment of resonances in the fluorine-19 nuclear magnetic resonance spectra of 5-fluorouracil-substituted transfer RNAs

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Abstract
Features of the 19F nuclear magnetic resonance (NMR) spectra of three purified 5-fluorouracil-(FUra-) substituted Escherichia coli tRNAs, tRNA1Val, tRNAmMet, and tRNAfMet, are compared. Each of the tRNA species can be resolved into two isoaccepting forms, A and B, whose 19F 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 tRNA1Val to FUra17 and the resonance at 4.6 ppm in the spectrum of fluorouracil-substituted tRNAmMet to FUra20. A reciprocal 19F{19F} nuclear Overhauser effect is observed between the downfield peaks A and B in the 19F NMR spectrum of 19F-labeled tRNA1Val. Assuming that fluorine-labeled tRNA1Val has a structure similar to that of yeast tRNAPhe, only FUra54 and -55 are close enough (4-5 .ANG.) to give an appreciable 19F homonuclear Overhauser effect. Peaks A and B have therefore been assigned to FUra54 and -55. As the temperature is raised from 30 to 45.degree.C, the intensity of peak B (6.6 ppm) in the spectrum of 19F-labeled tRNA1Val gradually shifts upfield to 6.4 ppm (Tm = 36.degree.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 methyl 1H and 13C signals in the spectra of several native tRNAs [Kastrup, R. V., and Schmidt, P. G. (1978) Nucleic Acids Res. 5, 257-269; Kopper, R. A., Schmidt, P. G., and Agris, P. F. (1983) Biochemistry 22, 1396-1401], we assign peak B to FUra54. Peak A in the spectrum of 19F-labeled tRNA1Val can then be assigned to FUra55. The lowest field resonance (peak A) in the 19F 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 the 19F NMR spectra of all three fluorinated tRNAs to the 5-fluorouracil residue that replaces the invariant .PSI. 55.