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

Abstract

Escherichia coli initiator methionine tRNA labeled in vivo with 5-fluorouracil (FUra) has been 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 diethylaminoethylcellulose (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 two FUra-substituted tRNAfMet-iso-accepting species, each with a specific activity over 1500 pmol/A260. Kinetic analysis shows both are aminoacylated at the same rate; apparent KmS for the two are 0.92 and 0.94 microM, compared with 1.7 microM for normal tRNAfMet. Chromatographic differences between the two forms of fluorinated tRNAfMet persist after aminoacylation, and the two tRNAs 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 two methionine tRNAs shows that they differ in only one oligonucleotide. The sequence 20FpApGp, derived from the dihydrouridine loop and stem region, which is found in one 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 two 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 one 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.

Noninvasive and quantitative 19F nuclear magnetic resonance study of flucytosine metabolism in Candida strains

“…In Escherichia coli cells, the RNA-incorporated FUTP gives a very broad 19F NMR signal which is located between 1 and 10 ppm downfield from 5FU (7,9); this resonance region also corresponds to that of Fns and Fnt. In tumor cells, FdUMP forms a ternary covalent complex with the enzyme TS and with the cofactor 5,10-methylenetetrahydrofolate (8); the native complex prepared in vitro gives a broad 19F NMR resonance at 8 = -102.1 ppm from external CF3COOH (3).…”

Fluorine-19 nuclear magnetic resonance study of codonanticodon interaction in 5-fluorouracil-substitutedE. colitransfer RNAs

“…5-Fluorouracil-substituted tRNAs were isolated from E. coli B cells grown in 5-fluorouracil-containing medium (20), by slight modification of the method of Hills et al (17). Amino acid accepting activity (20) was greater than 1.4 nmol per A260 unit for all tRNA preparations.…”

“…sequence found in the T-loop of most tRNAs. In the fluorinated tRNAs the TPPCP G sequence is replaced by FpFpCpG (17,20), and binding of CpGpApA should perturb the 19F resonances of fluorouracil residues 54 and 55. Figure 5 shows the 19F NMR spectrum of (FUra)tRNAyal before (A) and after (B) the addition of a 3-fold molar excess of CpGpApA.…”

“…Recent results have shown that 19F NMR spectroscopy of 5-fluorouracil (FUra)-substituted tRNAs is a sensitive method for probing the solution structure of tRNA (16)(17)(18)(19). The 19F-labeled tRNAs represent ideal subjects for investigation by 19F NMR because they remain fully active in protein synthesis in vitro despite having essentially all their uracil and uracilderived modified bases replaced by 5-fluorouracil (20)(21)(22)(23).…”

“…19F NMR spectra of the three FUra-substituted E. coli tRNAs thus far purified, tRNAyal (16,24), tRNApet (17), and tRNAMet (25), show resolved 19F resonances for virtually every incorporated fluorouracil. Each tRNA has two FUra residues in the anticodon loop.…”