In Vitro Biosynthesis of Pseudouridine at the Polynucleotide Level by an Enzyme Extract from Escherichia coli

DNA from Mycoplasma sp. Kid which was enriched for tRNA genes (containing about 10% tDNA) was transcribed by E. coli RNA polymerase. The RNA transcription product labeled with [(14)C]uridine was formed in good yield (70-fold net synthesis). After incubation of this [(14)C]uridine-labeled RNA with E. coli extracts, nucleotide analyses revealed that [(14)C]pseudouridine was formed. The experiments support the idea that the conversion of uridine to pseudouridine takes place at the macromolecular level. Furthermore, the conversion was shown to be specific for a uridine residue in tRNA-like material since neither [(14)C]polyuridylic acid nor the [(14)C]uridine-labeled RNA transcribed from lambda DNA served as substrate for the pseudouridine-forming enzyme(s).