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).
D001345:Autoradiography; D001711:Biotransformation; D002247:Carbon Isotopes; D002499:Centrifugation, Density Gradient; D002855:Chromatography, Thin Layer; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000201:enzymology*; D005815:Genetic Code; D066298:In Vitro Techniques; D009174:Mycoplasma; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D012329:RNA, Bacterial / Q000096:biosynthesis; D012343:RNA, Transfer / Q000096:biosynthesis; D014500:Uracil Nucleotides; D014529:Uridine / Q000096:biosynthesis*
D002247:Carbon Isotopes; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D014500:Uracil Nucleotides; D012343:RNA, Transfer; D012316:RNA Nucleotidyltransferases; D014529:Uridine