Structural studies of sigma54-mediated transcription initiation

转录是所有生物体基因表达的重要步骤，涉及到RNA聚合酶将序列信息从基因组DNA转化为单链RNA。转录起始是转录过程中最受调控的一步，对于确保生物体适应和响应环境变化并维持体内平衡至关重要。在细菌中，转录起始是由0个因子介导的，这些因子被分类为管家o70家族或主要变异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聚合酶融化并将DNA装载到其活性位点以形成合格的开放复合物的能力。o54启动子与营养消耗和应激反应有关，o54的独特之处在于它可以招募RNA聚合酶形成稳定的封闭复合物。o54最初通过阻断DNA装载来抑制开放复合物的形成，并占据活性位点并位于RNA出口通道的顶部。因此，开放复合物的形成需要一个特殊的分支6 AAA+（与多种细胞活性相关的ATP酶）蛋白家族的ATP水解，称为细菌增强子结合蛋白。此外，o54必须经历一系列构象变化，以使启动子逃逸并过渡到过程延伸。利用生物化学方法结合低温电子显微镜，我们捕获了多个AAA+参与的中间状态，以阐明开放复合物形成过程中早期DNA融化的机制。此外，我们已经确定了捕获转录初始阶段的高分辨率结构，从而深入了解o54在启动子逃逸过程中是如何移位的。这项工作加深了我们对o54介导的转录起始的理解，证明了所有细菌中启动子逃逸的保守机制，并证明了一个特殊的AAA+蛋白家族的独特转录激活机制。

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.