In vitro analysis of elongation and termination by mutant RNA polymerases with altered termination behavior.
In vitro analysis of elongation and termination by mutant RNA polymerases with altered termination behavior.
复制标题
终止行为改变的突变 RNA 聚合酶的延伸和终止的体外分析。
DOI：
10.1128/mcb.16.11.6468
复制
发表时间：
1996
影响因子：
5.3
通讯作者：
Hall,BD
中科院分区：
文献类型：
--
作者：
Shaaban,SA;Bobkova,EV;Chudzik,DM;Hall,BD
我们研究了几种酵母RNA聚合酶III(PolIII)突变酶的体外伸长和终止特性，这些酶在体内改变了终止行为(S.A.Shaaban，B.M.Krupp和B.D.Hall，Mol.牢房。比奥尔。15：1467-1478,1995)。对三种突变酶的完整转录本释放模式也进行了表征。所研究的突变占据了RET1蛋白的300到325、455到521和1061到1082氨基酸区域(P.James，S.Whelen和B.D.Hall，J.Biol。化学。266：5616-5624)，酵母RNA PolIII的第二大亚基。一般来说，末端增加的突变酶需要比野生型PolIII更长的时间来穿过模板基因；相反，对于大多数末端减少的突变体也是如此。与野生型酶相比，1个末端增加突变体(K310T I324K)速度更快，2个末端末端减少突变体(K512N和T455I E478K)更慢。在大多数情况下，总伸长动力学的这些变化可以通过相应地在SUP4tRNATYr基因内的停顿位置停留更长或更短的时间来解释。在分析的三个突变体中，一个突变体(T455I)与野生型相似，而另外两个突变体(T455I E478K和E478K)与完整的SUP4pre-tRNA结合更强烈。这项研究的结果支持终止是一个多步骤的途径，其中的几个不同区域的RET1蛋白积极参与。区域300到325可能影响RNA释放的一个步骤，而Rif同源区，氨基酸455到521，与新生的RNA 3‘端相互作用。几个突变对伸长动力学和RNA释放的双重影响表明，受它们影响的蛋白质基序在导致转录终止的步骤中具有多种作用。
We have studied the in vitro elongation and termination properties of several yeast RNA polymerase III (pol III) mutant enzymes that have altered in vivo termination behavior (S. A. Shaaban, B. M. Krupp, and B. D. Hall, Mol. Cell. Biol. 15:1467–1478, 1995). The pattern of completed-transcript release was also characterized for three of the mutant enzymes. The mutations studied occupy amino acid regions 300 to 325, 455 to 521, and 1061 to 1082 of the RET1 protein (P. James, S. Whelen, and B. D. Hall, J. Biol. Chem. 266:5616–5624, 1991), the second largest subunit of yeast RNA pol III. In general, mutant enzymes which have increased termination require a longer time to traverse a template gene than does wild-type pol III; the converse holds true for most decreased-termination mutants. One increased-termination mutant (K310T I324K) was faster and two reduced termination mutants (K512N and T455I E478K) were slower than the wild-type enzyme. In most cases, these changes in overall elongation kinetics can be accounted for by a correspondingly longer or shorter dwell time at pause sites within theSUP4tRNATyrgene. Of the three mutants analyzed for RNA release, one (T455I) was similar to the wild type while the two others (T455I E478K and E478K) bound the completedSUP4pre-tRNA more avidly. The results of this study support the view that termination is a multistep pathway in which several different regions of the RET1 protein are actively involved. Region 300 to 325 likely affects a step involved in RNA release, while the Rif homology region, amino acids 455 to 521, interacts with the nascent RNA 3' end. The dual effects of several mutations on both elongation kinetics and RNA release suggest that the protein motifs affected by them have multiple roles in the steps leading to transcription termination.