Isolation and characterization of two 5-fluorouracil-substituted Escherichia coli initiator methionine transfer ribonucleic acids

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Abstract
E. coli initiator methionine tRNA (tRNAfMet) labeled in vivo with 5-fluorouracil (FUra) was isolated and characterized. The tRNA, with essentially all its uracil and uracil-derived minor bases replaced by FUra, was purified by sequential chromatography, first on DEAE-cellulose at pH 8.9, followed by chromatography on Sepharose 4B using a reverse salt gradient, then on DEAE-Sephadex A-50, and finally on benzoylated DEAE-cellulose. The last step resolved 2 FUra-substituted tRNAfMet-isoaccepting species, each with a specific activity > 1500 pmol/A260. Kinetic analysis shows both are aminoacylated at the same rate; apparent Km for the 2 are 0.92 and 0.94 .mu.M, compared with 1.7 .mu.M for normal tRNAfMet. Chromatographic differnces between the 2 forms of fluorinated tRNAfMet persist after aminoacylation, and the 2 tRNA are not interconverted by denaturation and renaturation. The isoacceptors have nearly identical nucleoside composition and both contain 7-methylguanosine and 2''-O-methylcytidine as the only modified nucleosides. Analysis of complete RNase T1 digests of the 2 methionine tRNA shows that they differ in only 1 oligonucleotide. The sequence 20FpApGp, derived from the dihydrouridine loop and stem region, which is found in 1 of the isoaccepting forms of the tRNA, is replaced by an oligonucleotide containing adenine and guanine, but no FUra in the other. A modified FUra, with the properties of a 5-fluoro-5,6-dihydrouracil derivative, is detected in this tRNA. 19F NMR spectra of the 2 species of FUra-substituted initiator tRNA show 9-10 resolved resonances for the 12 FUra residues incorporated. The spectra differ primarily in the shift of 1 peak in the form lacking the sequence 20FpApGp, from 4.8 ppm downfield from free FUra (=0 ppm) to 14.9 ppm upfield from the standard.