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.

Photoaffinity labeling of catechol O-methyltransferase with 8-azido-S-adenosylmethionine.

An in vitro system using an enzyme extract containing ATP:L-methionine S-adenosyltransferase from Escherichia coli MRE 600 cells was used to synthesize 8-azido-S-adenosyl-L-methionine from methionine and 8-azidoadenosine 5'-triphosphate. In the absence of ultraviolet light and analog can serve as a methyl donor for porcine catechol O-methyltransferase. Photolysis of 8-azido-S-adenosyl[35S]methionine in the presence of catechol O-methyltransferase results in covalent incorporation. Addition of either authentic S-adenosylmethionine or S-adenosylhomocysteine, but not adenosine 5'-monophosphate, to the photolysis reaction mixture eliminates the photoincorporation. These results indicate that the incorporation is occurring at the S-adenosylmethionine binding site in the catechol O-methyltransferase.

Loss of tRNA 5-methyluridine methyltransferase and pseudouridine synthetase activities in 5-fluorouracil and 1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur)-treated Escherichia coli.

Transfer RNAs from Escherichia coli B treated with either 5-fluorouracil or its analog, 1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur), contain low levels of 5-fluorouracil, but are grossly deficient in pseudouridine and 5-methyluridine. The enzymes responsible for the formation of these two modified nucleosides, tRNA pseudouridine synthetase and (5-methyluridine)-methyltransferase, show substantially reduced activity levels in extracts from ftorafur- and 5-fluorouracil-treated cells relative to preparations from normal cells. When these tRNA-modifying activities are examined in vitro, both are inhibited by the addition of fluorouridine-containing tRNAs to the reaction mixtures. Pseudouridine synthetase activity shows potent inhibition. These inhibitory properties of fluorouridine-containing tRNAs, plus the inability of tRNA (5-methyluridine)-methyl-transferase to efficiently use fluorouridine-containing tRNAs as substrates, appear to account for the deficiency of 5-methyluridine and pseudouridine in tRNAs from cells containing low levels of 5-fluorouracil.

Nucleoside changes in tRNAs from Bacillus subtilus treated with 5-fluorouracil.

Extensive amounts of 5-fluorouridine and lower levels of 5-fluorocytidine are incorporated into tRNAs from Bacillus subtilus grown in the presence of 5-fluorouracil. Nucleoside analyses revealed both pseudouridine and 5-methyl-uridine levels to be reduced more extensively at low levels of analog incorporation than could be accounted for by a stoichiometric replacement, as observed earlier with Escherichia coli.