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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase
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Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase

机译：大肠杆菌RNA聚合酶在开放复合物形成的第一个动力学上重要的中间体中的DNA实时足迹

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摘要

The architecture of cellular RNA polymerases (RNAPs) dictates that transcription can begin only after promoter DNA bends into a deep channel and the start site nucleotide (+1) binds in the active site located on the channel floor. Formation of this transcriptionally competent "open" complex (RP_o) by Escherichia coli RNAP at the λP_R promoter is greatly accelerated by DNA upstream of base pair -47 (with respect to +1). Here we report real-time hydroxyl radical (·OH) and potassium permanganate (KMnO_4)) footprints obtained under conditions selected for optimal characterization of the first kinetically significant intermediate (I_1) in RP_o formation. ·OH footprints reveal that the DNA backbone from -71 to -81 is engulfed by RNAP in I_1 but not in RP_o; downstream protection extends to approximately +20 in both complexes. KMnO_4 footprinting detects solvent-accessible thymine bases in RP_o, but not in I_1. We conclude that upstream DNA wraps more extensively on RNAP in I_1 than in RP_o and that downstream DNA (-11 to +20) occupies the active-site channel in I_1 but is not yet melted. Mapping of the footprinting data onto available x-ray structures provides a detailed model of a kinetic intermediate in bacterial transcription initiation and suggests how transient contacts with upstream DNA in I_1 might rearrange the channel to favor entry of downstream duplex DNA.
机译：细胞RNA聚合酶（RNAP）的结构要求转录只能在启动子DNA弯曲进入深通道并且起始位点核苷酸（+1）结合在通道底的活性位点后才能开始。大肠杆菌RNAP在λP_R启动子上形成这种转录感受态“开放”复合物（RP_o），在碱基对-47的上游（相对于+1）大大促进了DNA的形成。在这里，我们报告了实时羟基自由基（·OH）和高锰酸钾（KMnO_4））的足迹，这些足迹是在为RP_o形成中第一个具有动力学意义的中间体（I_1）的最佳表征选择的条件下获得的。 ·OH足迹表明，I_1中的RNAP吞噬了-71至-81的DNA骨架，而RP_o中则没有。在两个联合体中，下游保护均扩展至大约+20。 KMnO_4占用空间可检测RP_o中可访问溶剂的胸腺嘧啶碱基，但不适用于I_1。我们得出的结论是，上游DNA在I_1中的RNAP上比RP_o中的包裹更广泛，并且下游DNA（-11至+20）在I_1中占据了活性位点通道，但尚未融化。将足迹数据映射到可用的X射线结构上，提供了细菌转录起始过程中动力学中间体的详细模型，并提出了与I_1中上游DNA的瞬时接触如何重新排列通道，从而有利于下游双链DNA的进入。

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