Mekler Vladimir, Minakhin Leonid, Borukhov Sergei, Mustaev Arkady, Severinov Konstantin
Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, NJ 08854, USA.
Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, NJ 08854, USA.
J Mol Biol. 2014 Dec 12;426(24):3973-3984. doi: 10.1016/j.jmb.2014.10.005. Epub 2014 Oct 13.
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.
细菌RNA聚合酶（RNAP）在起始复合物和延伸复合物中与转录泡下游的双链DNA广泛接触。我们通过测量稳定的RNAP复合物与模型启动子DNA片段的起始活性，研究了下游相互作用在形成具有催化活性的转录起始复合物中的作用，这些模型启动子DNA片段的下游末端相对于转录起始位点（+1）从+3延伸到+21。我们发现，当转录起始位点上游的RNAP-启动子相互作用很强且启动子解链区域富含AT时，+6位置下游的DNA在转录起始中不起重要作用。进一步缩短下游DNA会显著降低转录起始效率。随着启动子解链区域GC含量的增加或在细菌严谨反应调节因子DksA和ppGpp存在的情况下，有效转录起始所需的最小下游DNA双链的边界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才能保持起始复合物的催化活性构象，并且RNAP与下游DNA之间的接触建立可以与启动子解链相偶联。数据进一步表明，RNAP与转录泡紧邻下游的DNA相互作用对于转录起始尤为重要。我们推测，这些靠近活性中心的接触稳定了活性中心裂隙中的DNA模板链和/或定位了RNAP钳结构域以允许RNA合成。
