Naji Souad, Bertero Michela G, Spitalny Patrizia, Cramer Patrick, Thomm Michael
Lehrstuhl für Mikrobiologie und Archaeenzentrum, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany.
Nucleic Acids Res. 2008 Feb;36(2):676-87. doi: 10.1093/nar/gkm1086. Epub 2007 Dec 10.
The active center clefts of RNA polymerase (RNAP) from the archaeon Pyrococcus furiosus (Pfu) and of yeast RNAP II are nearly identical, including four protruding loops, the lid, rudder, fork 1 and fork 2. Here we present a structure-function analysis of recombinant Pfu RNAP variants lacking these cleft loops, and analyze the function of each loop at different stages of the transcription cycle. All cleft loops except fork 1 were required for promoter-directed transcription and efficient elongation. Unprimed de novo transcription required fork 2, the lid was necessary for primed initial transcription. Analysis of templates containing a pre-melted bubble showed that rewinding of upstream DNA drives RNA separation from the template. During elongation, downstream DNA strand separation required template strand binding to an invariant arginine in switch 2, and apparently interaction of an invariant arginine in fork 2 with the non-template strand.
嗜热栖热菌（Pfu）的RNA聚合酶（RNAP）与酵母RNAP II的活性中心裂隙几乎相同，包括四个突出的环，即盖子、舵、叉1和叉2。在这里，我们展示了缺乏这些裂隙环的重组Pfu RNAP变体的结构-功能分析，并分析了每个环在转录周期不同阶段的功能。除叉1外，所有裂隙环都是启动子导向转录和有效延伸所必需的。无引物的从头转录需要叉2，盖子是引物起始转录所必需的。对含有预解链泡的模板的分析表明，上游DNA的重新缠绕驱动RNA与模板分离。在延伸过程中，下游DNA链的分离需要模板链与开关2中的一个不变精氨酸结合，并且显然叉2中的一个不变精氨酸与非模板链相互作用。
