We have previously purified and characterized two different S. cerevisiae enzymes that produce pseudouridine specifically in nucleotide positions 13 and 55, respectively, in their tRNA substrates. The interactions of these enzymes with fluorinated tRNAs have now been studied. Such RNAs were produced by in vitro transcription using as templates synthetic genes that encode variants of a yeast glycine tRNA. RNAs substituted with fluorouracil were found to markedly inhibit pseudouridine synthase activity and the inhibitory effect of a tRNA was to a large extent dependent on the presence of fluorouracil in the nucleotide position where normally pseudouridylation occurs. Pseudouridine synthases were shown to form highly stable, non-covalent complexes with fluorinated tRNAs and we demonstrate that this interaction may be used to further characterize and purify these enzymes. The use of 5-fluorouracil as a cancer therapeutic agent is discussed in relation to our results.
Base Sequence Chromatography, Liquid