PMID:3276686
| Citation |
Kammen, HO, Marvel, CC, Hardy, L and Penhoet, EE (1988) Purification, structure, and properties of Escherichia coli tRNA pseudouridine synthase I. J. Biol. Chem. 263:2255-63 |
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| Abstract |
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. |
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| Keywords |
Amino Acid Sequence; Escherichia coli/enzymology; Intramolecular Transferases; Isomerases/genetics; Isomerases/isolation & purification; Molecular Sequence Data; Molecular Weight; Nucleic Acid Conformation; Protein Biosynthesis; RNA, Transfer/metabolism; Transcription, Genetic |
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