Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia.
Nucleic Acids Res. 2012 Apr;40(7):3078-91. doi: 10.1093/nar/gkr1158. Epub 2011 Dec 2.
A transcription initiation factor, the σ(70) subunit of Escherichia coli RNA polymerase (RNAP) induces transcription pausing through the binding to a promoter-like pause-inducing sequence in the DNA template during transcription elongation. Here, we investigated the mechanism of σ-dependent pausing using reconstituted transcription elongation complexes which allowed highly efficient and precisely controlled pause formation. We demonstrated that, following engagement of the σ subunit to the pause site, RNAP continues RNA synthesis leading to formation of stressed elongation complexes, in which the nascent RNA remains resistant to Gre-induced cleavage while the transcription bubble and RNAP footprint on the DNA template extend in downstream direction, likely accompanied by DNA scrunching. The stressed complexes can then either break σ-mediated contacts and continue elongation or isomerize to a backtracked conformation. Suppressing of the RNAP backtracking decreases pausing and increases productive elongation. On the contrary, core RNAP mutations that impair RNAP interactions with the downstream part of the DNA template stimulate pausing, presumably by destabilizing the stressed complexes. We propose that interplay between DNA scrunching and RNAP backtracking may have an essential role in transcription pausing and its regulation in various systems.
转录起始因子，大肠杆菌 RNA 聚合酶（RNAP）的σ（70）亚基在转录延伸过程中通过与 DNA 模板中类似启动子的暂停诱导序列结合诱导转录暂停。在这里，我们使用重新组装的转录延伸复合物研究了σ依赖性暂停的机制，该复合物允许高效且精确控制暂停形成。我们证明，在σ亚基与暂停位点结合后，RNAP 继续进行 RNA 合成，导致形成应激延伸复合物，其中新生 RNA 仍然对 Gre 诱导的切割具有抗性，而转录泡和 RNAP 在 DNA 模板上的足迹在下游方向延伸，可能伴随着 DNA 卷曲。然后，应激复合物可以打破 σ 介导的接触并继续延伸，或者异构化为回溯构象。抑制 RNAP 回溯会减少暂停并增加生产性延伸。相反，核心 RNAP 突变会削弱 RNAP 与 DNA 模板下游部分的相互作用，从而刺激暂停，这可能是通过破坏应激复合物来实现的。我们提出，DNA 卷曲和 RNAP 回溯之间的相互作用可能在各种系统中的转录暂停及其调控中发挥重要作用。
