Purification, Structure and Properties of 'Escherichia coli' tRNA Pseudouridine Synthase 1

摘要

The RNA modification enzyme, trna pseudourine synthase I (PSUI) has been isolated in 95% purity from an Escherichia coli strain harboring multicopy plasmid with a 2.3kb insert for the hisT operon. Its molecular size, amino acid composition and N-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 if PSUI 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, non substrates and non substrates tRNAs and does not require a monovalent cation. The findings are consistent with a multi-step mechanism whereby PSUI first binds non-specifically, then forms transient covalent adducts with tRNA substrates. Keywords: Charts; Graphs; Tables(Data).