Cryo-EM structure of Escherichia coli σ70 RNA polymerase and promoter DNA complex revealed a role of σ non-conserved region during the open complex formation
Cryo-EM structure of Escherichia coli σ70 RNA polymerase and promoter DNA complex revealed a role of σ non-conserved region during the open complex formation
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DOI：
10.1074/jbc.ra118.002161
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发表时间：
2018-05-11
影响因子：
4.8
通讯作者：
Murakami, Katsuhiko S.
中科院分区：
文献类型：
--
作者：
Narayanan, Anoop;Vago, Frank S.;Murakami, Katsuhiko S.
基因表达的第一步是通过包括RNA聚合酶(RNAP)在内的转录机制将储存在DNA中的遗传信息转录成RNA。在大肠杆菌中，一个主要的西格玛(70)因子形成RNAP全酶来表达家务基因。Sigma(70)在保守区1.2和2.1之间以及Sigma非保守区(Sigma(NCR))之间有一个很大的插入，但其功能仍有待阐明。在这项研究中，我们分别在4.2和5.75埃分辨率下测定了大肠杆菌RNAP sigma(70)全酶及其与启动子DNA的复合体(开放复合体，RPO)的冷冻-EM结构，以揭示RNAP和DNA的天然构象。这里的RPO结构在-10元件的上游发现了sigma(NCR)和启动子DNA之间的相互作用，这在以前确定的大肠杆菌RNAP转录起始复合体(RPO+短RNA)结构中没有观察到，因为晶体堆积效应的抑制。氨基酸替换(R157A/R157E)破坏了Sigma(NCR)与DNA的相互作用，影响了转录起始点附近的DNA开放，从而降低了RNAP的转录活性。我们认为，Sigma(NCR)与DNA的相互作用在蛋白质细菌中是保守的，而在其他细菌中，RNAP用转录因子取代了它的作用。
First step of gene expression is transcribing the genetic information stored in DNA to RNA by the transcription machinery including RNA polymerase (RNAP). In Escherichia coli, a primary sigma(70) factor forms the RNAP holoenzyme to express housekeeping genes. The sigma(70) contains a large insertion between the conserved regions 1.2 and 2.1, the sigma non-conserved region (sigma(NCR)), but its function remains to be elucidated. In this study, we determined the cryo-EM structures of the E. coli RNAP sigma(70) holoenzyme and its complex with promoter DNA (open complex, RPo) at 4.2 and 5.75 angstrom resolutions, respectively, to reveal native conformations of RNAP and DNA. The RPo structure presented here found an interaction between the sigma(NCR) and promoter DNA just upstream of the -10 element, which was not observed in a previously determined E. coli RNAP transcription initiation complex (RPo plus short RNA) structure by X-ray crystallography because of restraint of crystal packing effects. Disruption of the sigma(NCR) and DNA interaction by the amino acid substitutions (R157A/R157E) influences the DNA opening around the transcription start site and therefore decreases the transcription activity of RNAP. We propose that the sigma(NCR) and DNA interaction is conserved in proteobacteria, and RNAP in other bacteria replaces its role with a transcription factor.