RNA polymerase (RNAP) recognizes promoter DNA through many interactions that determine specificity of transcription initiation. In addition to the dedicated transcription initiation σ factor in bacteria, the core enzyme of RNAP can also participate in promoter recognition. In particular, guanine residue at the +2 position (+2G) of the nontemplate DNA strand is bound in the CRE pocket formed by the RNAP β subunit. Here, we analyzed the role of these contacts in the process of promoter escape by RNAP by studying point mutations in the β subunit of Escherichia coli RNAP that disrupted these interactions. We found that the presence of +2G in the promoter slowed down the rate of promoter escape and increased proportion of inactive complexes. Amino acid substitutions in the CRE pocket decreased the promoter complex stability and changed the pattern of short RNA products synthesized during initiation, but did not significantly affect the rate of transition to elongation, regardless of the presence of +2G. Thus, the contacts of the CRE pocket with +2G do not make a significant contribution to the kinetics of promoter escape by RNAP, while the observed changes in the efficiency of abortive synthesis are not directly related to the rate of promoter escape.

中文翻译：

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启动子逃逸过程中大肠杆菌 RNA 聚合酶 CRE 区与非模板 DNA 相互作用的作用

RNA 聚合酶 (RNAP) 通过许多决定转录起始特异性的相互作用识别启动子 DNA。除了细菌中专门的转录起始σ因子外，RNAP的核心酶还可以参与启动子识别。特别是，非模板 DNA 链 +2 位置 (+2G) 的鸟嘌呤残基结合在由 RNAP β 亚基形成的 CRE 口袋中。在这里，我们通过研究破坏这些相互作用的大肠杆菌 RNAP β 亚基中的点突变，分析了这些接触在 RNAP 启动子逃逸过程中的作用。我们发现启动子中+2G 的存在减慢了启动子逃逸的速度并增加了无活性复合物的比例。CRE 口袋中的氨基酸取代降低了启动子复合物的稳定性并改变了起始过程中合成的短 RNA 产物的模式，但无论是否存在 +2G，都不会显着影响向延伸的转变速率。因此，CRE 口袋与 +2G 的接触对 RNAP 启动子逃逸的动力学没有显着贡献，而观察到的无效合成效率的变化与启动子逃逸率没有直接关系。