Specific incorporation of 5-fluorocytidine into Escherichia coli RNA.

RNAs isolated from Escherichia coli B grown in the presence of 5-fluorouracil have high levels of the analog replacing uridine and uridine-derived modified nucleosides. Cytidine has also been shown to be replaced in these RNAs by 5-fluorocytidine, a metabolic product of 5-fluorouracil, but to a considerably lesser extent. When 5-fluorocytidine is added to cultured of E. coli B little 5-fluorocytidine (0.20 mol%) is incorporated into cellular RNAs because of the active cytosine/cytidine deaminase activities. Addition of the cytidine deaminase inhibitor tetrahydrouridine (70 micrograms/ml) increases 5-fluorocytidine incorporation to about 3 mol% in tRNAs, but does not eliminate 5-fluorouridine incorporation. E. coli mutants lacking cytosine/cytidine deaminase activities are able to more than double the extent of 5-fluorocytidine incorporation into their transfer and ribosomal RNAs, replacing cytidine with no detectable 5-fluorouridine incorporation. Levels of 5-methyluridine, pseudouridine and dihydrouridine in tRNAs are not affected. These fluorocytidine-containing tRNAs show amino acid-accepting activities similar to control tRNAs. Fluorocytidine was found to be quite susceptible to deamination under alkaline conditions. Its conversion to primarily 5-fluorouridine follows pseudo-first-order reaction kinetics with a half-life of 10 h in 0.3 M KOH at 37 degrees C. This instability in alkali probably explains why 5-fluorocytidine was not found earlier in RNAs isolated from cells treated with 5-fluorouridine, since most early RNA hydrolyses were carried out in alkali. It may also explain the mild mutagenic properties observed in some systems following 5-fluorouridine treatment. Initial 19F-NMR measurements in fluorocytidine-containing tRNAs indicate that this modified tRNA may be useful in future structural studies of tRNAs and in probing tRNA-protein complexes.