Nano positioning system reveals the course of upstream and nontemplate DNA within the RNA polymerase II elongation complex
Nano positioning system reveals the course of upstream and nontemplate DNA within the RNA polymerase II elongation complex
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DOI：
10.1093/nar/gkp601
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发表时间：
2009-09-01
影响因子：
14.9
通讯作者：
Michaelis, Jens
中科院分区：
文献类型：
--
作者：
Andrecka, Joanna;Treutlein, Barbara;Michaelis, Jens
RNA聚合酶II（Pol II）延伸复合物（EC）的晶体学研究揭示了下游DNA和DNA-RNA杂交体的位置，但不是转录泡中的非模板DNA链和上游DNA双链体的过程。在这里，我们使用单分子荧光共振能量转移（smFRET）实验，以我们最近开发的纳米定位系统（Nano Positioning System）定位非模板和上游DNA。在得到的Pol II EC的完整模型中，在位置+2处非模板与模板链的分离涉及与叉环2的相互作用。非模板链通过Pol II叶上的β 10-β 11环，然后转向方向舵上方裂缝的另一侧。上游DNA双链体以近似直角离开进入的下游DNA，并从突起和夹具之间的裂缝发出。与已发表的数据的比较表明，完整的EC的架构是保守的从细菌到真核生物和上游DNA的重新定位在启动延长过渡。
Crystallographic studies of the RNA polymerase II (Pol II) elongation complex (EC) revealed the locations of downstream DNA and the DNA-RNA hybrid, but not the course of the nontemplate DNA strand in the transcription bubble and the upstream DNA duplex. Here we used single-molecule Fluorescence Resonance Energy Transfer (smFRET) experiments to locate nontemplate and upstream DNA with our recently developed Nano Positioning System (NPS). In the resulting complete model of the Pol II EC, separation of the nontemplate from the template strand at position +2 involves interaction with fork loop 2. The nontemplate strand passes loop beta 10-beta 11 on the Pol II lobe, and then turns to the other side of the cleft above the rudder. The upstream DNA duplex exits at an approximately right angle from the incoming downstream DNA, and emanates from the cleft between the protrusion and clamp. Comparison with published data suggests that the architecture of the complete EC is conserved from bacteria to eukaryotes and that upstream DNA is relocated during the initiation-elongation transition.