Purification, structure, and properties of Escherichia coli tRNA pseudouridine synthase I.
The RNA modification enzyme, tRNA pseudouridine synthase I has been isolated in 95% purity from an Escherichia coli strain harboring a multicopy plasmid with a 2.3-kilobase pair insert from the hisT operon. Its molecular size, amino acid composition, and amino-terminal sequence correspond to those predicted by the structure and expression of the hisT gene. Enzyme activity, as measured by a 3H release assay, is unaffected by pretreatment of tRNA pseudouridine synthase I with micrococcal nuclease and is optimized by the addition of a monovalent cation and thiol reductant. The activity is inhibited by all tRNA species tested, including substrates, modified tRNAs, nonsubstrates, or tRNAs containing 5-fluorouridine. Binding of tRNA pseudouridine synthase I occurs with both substrate and nonsubstrate tRNAs and does not require a monovalent cation. Our findings are consistent with a multistep mechanism whereby tRNA pseudouridine synthase I first binds nonspecifically and then forms transient covalent adducts with tRNA substrates. In the absence of other proteins, purified tRNA pseudouridine synthase I forms psi at all three modification sites known to be affected in hisT mutants. The 36.4-kDa polypeptide product of the gene adjacent to hisT, whose translation is linked to that of tRNA pseudouridine synthase I, is not a functional subunit for tRNA pseudouridine synthase I activity, nor is it a separate synthase acting at one of the three loci.
Kammen
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Naval Biosciences Laboratory, University of California, Berkeley 94720.
Marvel
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CC
Hardy
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Penhoet
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D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology*; D019751:Intramolecular Transferases; D007535:Isomerases / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000378:metabolism; D014158:Transcription, Genetic
D012343:RNA, Transfer; D007535:Isomerases; D019751:Intramolecular Transferases; C023199:tRNA-pseudouridine synthase I