A Critical Role of Downstream RNA Polymerase-Promoter Interactions in the Formation of Initiation Complex
A Critical Role of Downstream RNA Polymerase-Promoter Interactions in the Formation of Initiation Complex
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DOI：
10.1074/jbc.m111.247080
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发表时间：
2011-06-24
影响因子：
4.8
通讯作者：
Severinov, Konstantin
中科院分区：
文献类型：
--
作者：
Mekler, Vladimir;Minakhin, Leonid;Severinov, Konstantin
启动子在细菌中融化的成核与RNA聚合酶（RNAP）结合到位于转录泡上游边缘的保守的-10启动子元件上偶联。转录泡向下游传播以包括转录起始位点的机制尚不清楚。在这里，我们介绍了新的模型下游叉连接启动子片段，特异性结合RNAP和模仿下游段的启动子复合物。我们证明，RNAP结合到下游叉路口是与周围的转录起始点的DNA熔化耦合。因此，某些下游叉连接探针可以用作转录模板。使用蛋白质信标荧光方法，我们确定RNAP下游叉连接复合物的亲和力和转录活性的结构决定因素。RNAP与双链启动子片段的相互作用的测量揭示RNAP与下游DNA的相互作用的强度在启动子开放中起着关键作用，并且下游双链体的长度必须超过有效形成有转录能力的开放启动子复合物的临界长度。
Nucleation of promoter melting in bacteria is coupled with RNA polymerase (RNAP) binding to a conserved - 10 promoter element located at the upstream edge of the transcription bubble. The mechanism of downstream propagation of the transcription bubble to include the transcription start site is unclear. Here we introduce new model downstream fork junction promoter fragments that specifically bind RNAP and mimic the downstream segment of promoter complexes. We demonstrate that RNAP binding to downstream fork junctions is coupled with DNA melting around the transcription start point. Consequently, certain downstream fork junction probes can serve as transcription templates. Using a protein beacon fluorescent method, we identify structural determinants of affinity and transcription activity of RNAP-downstream fork junction complexes. Measurements of RNAP interaction with double-stranded promoter fragments reveal that the strength of RNAP interactions with downstream DNA plays a critical role in promoter opening and that the length of the downstream duplex must exceed a critical length for efficient formation of transcription competent open promoter complex.