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首页> 外文期刊>Journal of Molecular Biology >Coupling of Downstream RNA Polymerase-Promoter Interactions with Formation of Catalytically Competent Transcription Initiation Complex
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Coupling of Downstream RNA Polymerase-Promoter Interactions with Formation of Catalytically Competent Transcription Initiation Complex

机译：下游RNA聚合酶 - 促进剂相互作用与催化竞争性转录起始复合体的形成

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摘要

Bacterial RNA polymerase (RNAP) makes extensive contacts with duplex DNA downstream of the transcription bubble in initiation and elongation complexes. We investigated the role of downstream interactions in formation of catalytically competent transcription initiation complex by measuring initiation activity of stable RNAP complexes with model promoter DNA fragments whose downstream ends extend from +3 to +21 relative to the transcription start site at +1. We found that DNA downstream of position +6 does not play a significant role in transcription initiation when RNAP-promoter interactions upstream of the transcription start site are strong and promoter melting region is AT rich. Further shortening of downstream DNA dramatically reduces efficiency of transcription initiation. The boundary of minimal downstream DNA duplex needed for efficient transcription initiation shifted further away from the catalytic center upon increasing the GC content of promoter melting region or in the presence of bacterial stringent response regulators DksA and ppGpp. These results indicate that the strength of RNAP-downstream DNA interactions has to reach a certain threshold to retain the catalytically competent conformation of the initiation complex and that establishment of contacts between RNAP and downstream DNA can be coupled with promoter melting. The data further suggest that RNAP interactions with DNA immediately downstream of the transcription bubble are particularly important for initiation of transcription. We hypothesize that these active center-proximal contacts stabilize the DNA template strand in the active center cleft and/or position the RNAP clamp domain to allow RNA synthesis. (C) 2014 Elsevier Ltd. All rights reserved.
机译：细菌RNA聚合酶（RNAP）在起始和伸长复合物中与转录泡下游的双相DNA进行广泛的接触。通过测量稳定的RNAP复合物的起始活性与模型启动子DNA片段的起始活性来调查下游相互作用在催化竞技转录开始复合物的形成中的作用，其下游末端相对于+1的转录开始点从+ 3- + 21延伸。我们发现，当转录开始点上游的RNAP启动子相互作用是强大的，致力于融化区域时，位于+6下游的DNA在转录开始中发挥显着作用。进一步缩短下游DNA显着降低了转录起始的效率。在增加启动子熔化区域的GC含量或在细菌严格响应调节剂DKSA和PPGPP存在下，有效转录起始所需的最小下游DNA双链体进一步远离催化中心。这些结果表明，RNAP下游DNA相互作用的强度必须达到一定阈值以保持启动复合物的催化竞争性构象，并且在RNAP和下游DNA之间建立触点可以与启动子熔化偶联。数据进一步表明，与转录泡沫的下游的与DNA的RNAP相互作用对于开始转录尤为重要。我们假设这些活性中心近端触点稳定在活性中心裂缝中的DNA模板链和/或定位RNAP钳位结构域以允许RNA合成。 （c）2014年elestvier有限公司保留所有权利。

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