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 [¹⁴C]uridine was formed in good yield (70-fold net synthesis). After incubation of this [¹⁴C]uridine-labeled RNA with E. coli extracts, nucleotide analyses revealed that [¹⁴C]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 [¹⁴C]polyuridylic acid nor the [¹⁴C]uridine-labeled RNA transcribed from λ DNA served as substrate for the pseudouridine-forming enzyme(s).