Purification and properties of a mammalian tRNA pseudouridine synthase.
A tRNA pseudouridine synthase has been extensively purified from steer thymus extracts, using undermodified tRNA from hisT- mutants of Salmonella typhimurium as a substrate. The enzyme synthesizes a group of psi residues in the anticodon region of various hisT- isoacceptors and behaves like a eukaryotic homologue of Salmonella tRNA psi synthase I. The thymus enzyme requires a thiol and a monovalent cation (NH4+ or K+) for optimum activity; no energy sources or cofactors are required. The activity is inhibited by single tRNAs or bulk tRNA from all sources tested, and by ribosomal RNAs, various polyribonucleotides, and DNA. The enzyme modifies the two hisT- tRNAPhe isoacceptors, both tRNATyr acceptors and at least five of the tRNALeu isoacceptors to products that coelute with the respective wild type species on reverse-phase columns. With pure hisT- tRNA2Phe as substrate, the enzyme specifically converts residue U39 to psi. Interestingly, a psi residue is still present at position 32, in the anticodon loop of hisT- tRNA2Phe, indicating the existence of other uncharacterized pseudouridylation enzymes in S. typhimurium. These composite results show that the thymus enzyme can form psi at residues 38, 39, and 40 in the anticodon region of appropriate hisT- isoacceptors. During the enzyme purification, a second activity is partially resolved, which releases 3H from wild type S. typhimurium [pyrimidine-5-3H]tRNA. This activity may be associated with an enzyme that pseudouridylates sites that are uniquely modified in eukaryotic tRNAs, but not in Salmonella tRNAs. Our observations support the view that the psi residues in tRNA are synthesized by a family of enzymes, whose members act on uridine residues in specific regions of the molecule.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D019751:Intramolecular Transferases; D007535:Isomerases / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008297:Male; D009690:Nucleic Acid Conformation; D011560:Pseudouridine / Q000302:isolation & purification / Q000378:metabolism; D012343:RNA, Transfer / Q000302:isolation & purification / Q000378:metabolism; D012486:Salmonella typhimurium; D013950:Thymus Gland / Q000201:enzymology*
D011560:Pseudouridine; D012343:RNA, Transfer; D007535:Isomerases; D019751:Intramolecular Transferases; C023199:tRNA-pseudouridine synthase I; D000604:Amino Acyl-tRNA Synthetases