Discontinuous movement and conformational change during pausing and termination by T7 RNA polymerase.
Discontinuous movement and conformational change during pausing and termination by T7 RNA polymerase.
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T7 RNA 聚合酶暂停和终止期间的不连续运动和构象变化。
DOI：
10.1093/emboj/cdg618
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Sousa,Rui
中科院分区：
文献类型：
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作者：
Mukherjee,Srabani;Brieba,LuisG;Sousa,Rui
使用位点特异性束缚的化学核酸酶对 T7 RNA 聚合酶在 II 类终止位点暂停和终止的时间分辨表征进行了研究。数据表明 T7RNAP 通常作为刚体沿着模板均匀移动。然而，在 II 类位点，这种运动被中断，聚合酶的前缘沿着 DNA 比后缘移动得更远。这种不连续的运动可能会持续下去，直到它不再能够被延伸复合物中的构象变化所适应，此时聚合酶可以暂停或终止。通过在聚合酶指和拇指子结构域之间引入二硫键来消除终止，但不是暂停。引入的半胱氨酸破坏了拇指-手指盐桥，并且在还原条件下，这种突变酶显示出持续合成能力降低，同时 RNA 延伸至 5 nt。这些观察结果表明，终止需要拇指和手指子域分开，从而逆转对于最初形成稳定转录复合物很重要的构象变化。
Time‐resolved characterization of T7 RNA polymerase pausing and terminating at a class II termination site has been carried out using site‐specifically tethered chemical nucleases. The data indicate that T7RNAP normally moves uniformly down the template as a rigid body. However, at the class II site this movement is interrupted, and the leading edge of the polymerase moves further along the DNA than the trailing edge. This discontinuous movement may persist until it can no longer be accommodated by conformational changes in the elongation complex, at which point the polymerase can either pause or terminate. Termination, but not pausing, is abrogated by introduction of a disulfide bond between the polymerase fingers and thumb subdomains. The introduced cysteines disrupt a thumb–fingers salt‐bridge and, under reducing conditions, this mutant enzyme shows reduced processivity coincident with extension of the RNA to 5 nt. These observations suggest that termination requires that the thumb and fingers subdomains move apart, in a reversal of a conformational change important for initially forming a stable transcription complex.