Kammen H O, Marvel C C, Hardy L, Penhoet E E
Naval Biosciences Laboratory, University of California, Berkeley 94720.
J Biol Chem. 1988 Feb 15;263(5):2255-63.
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.
RNA修饰酶——tRNA假尿苷合酶I已从携带含有来自hisT操纵子的2.3千碱基对插入片段的多拷贝质粒的大肠杆菌菌株中分离出来，纯度达95%。其分子大小、氨基酸组成和氨基末端序列与hisT基因的结构和表达所预测的一致。通过3H释放测定法测得的酶活性不受微球菌核酸酶预处理tRNA假尿苷合酶I的影响，并且通过添加一价阳离子和硫醇还原剂可使其活性达到最佳。该活性受到所有测试的tRNA种类的抑制，包括底物、修饰的tRNA、非底物或含有5-氟尿苷的tRNA。tRNA假尿苷合酶I与底物和非底物tRNA均发生结合，且不需要一价阳离子。我们的研究结果与一种多步骤机制一致，即tRNA假尿苷合酶I首先非特异性结合，然后与tRNA底物形成瞬时共价加合物。在没有其他蛋白质的情况下，纯化的tRNA假尿苷合酶I在hisT突变体中已知受影响的所有三个修饰位点形成假尿苷。与hisT相邻的基因的36.4 kDa多肽产物，其翻译与tRNA假尿苷合酶I的翻译相关联，它既不是tRNA假尿苷合酶I活性的功能亚基，也不是在三个位点之一起作用的单独合酶。
