Structural studies of sigma54-mediated transcription initiation

转录是所有生物基因表达中必不可少的步骤，它涉及到由RNA聚合酶将序列信息从基因组DNA转化为单链RNA的过程。转录启动是转录过程中最受调控的步骤，对于确保生物体能够适应和响应环境的变化并保持动态平衡是必不可少的。在细菌中，转录起始是由o因子介导的，o因子被归类为持家因子o70家族或主要变体o54。O因子将RNA聚合酶招募到基因的上游启动子区域，并调节RNA聚合酶融化DNA并将DNA加载到其活性部位形成合格的开放复合体的能力。O54启动子与营养耗竭和应激反应有关，而O54是独一无二的，因为它招募RNA聚合酶形成稳定的封闭复合体。O54最初通过阻止DNA装载以及占据活性部位和坐在RNA出口通道的顶部来抑制开放复合体的形成。因此，开放复合体的形成需要被称为细菌增强子结合蛋白的一种特殊的支链6 AAA+(与不同细胞活动相关的ATPase)蛋白家族的ATP水解。此外，o54还必须经历一系列构象变化才能使启动子逃逸和向过程延伸过渡。使用生化方法结合冷冻电子显微镜，我们捕获了多个AAA+参与的中间态，以阐明开放复合体形成过程中早期DNA熔化的机制。此外，我们还确定了捕捉转录初始阶段的高分辨率结构，从而深入了解了o54在启动子逃逸过程中是如何移位的。本文的工作加深了我们对o54介导的转录启动的理解，展示了所有细菌中启动子逃逸的保守机制，并展示了一个特殊的AAA+蛋白家族激活转录的独特机制。

Transcription is an essential step in gene expression in all organisms and involves the conversion of sequential information from genomic DNA to single-stranded RNA by the enzyme RNA polymerase. Transcription initiation is the most regulated step in transcription and is essential in ensuring an organism can adapt and respond to changes in its environment and maintain homeostasis. In bacteria, transcription initiation is mediated by o factors, which are classified as housekeeping o70 family or major variant o54. o factors recruit RNA polymerase to upstream promoter regions of genes and regulate the ability for RNA polymerase to melt and load DNA into its active site to form a competent open complex.o54 promoters are associated with nutrient depletion and stress response, and o54 is unique as it recruits RNA polymerase to form a stable closed complex. o54 initially inhibits open complex formation by blocking DNA loading, as well as occupying the active site and sitting atop of the RNA exit channel. Open complex formation therefore requires ATP hydrolysis by a specialised family of clade 6 AAA+ (ATPase associated with diverse cellular activity) proteins called bacterial enhancer binding proteins. Furthermore, o54 must undergo a set of conformational changes to enable promoter escape and a transition to processive elongation.Using biochemical approaches coupled with cryo-electron microscopy, we captured multiple AAA+-engaged intermediate states to elucidate the mechanism of early DNA melting during open complex formation. Furthermore, we have determined high-resolution structures capturing the initial stages of transcription, gaining insights into how o54 is displaced during promoter escape.The work presented here deepens our understanding of o54 mediated transcription initiation, demonstrates conserved mechanisms of promoter escape in all bacteria and demonstrates a unique mechanism of transcription activation by a specialised family of AAA+ protein.