Structural studies of sigma54-mediated transcription initiation

sigma54 介导的转录起始的结构研究

• 批准号: 2368496
• 金额: --
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2368496
• 关键词: Structural; studies; sigma54; mediated; transcription

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.

转录是所有生物体中基因表达的重要步骤，涉及通过RNA聚合酶将序列信息从基因组DNA转化为单链RNA。转录起始是转录中最受调控的步骤，对于确保生物体能够适应和响应其环境的变化并维持稳态至关重要。在细菌中，转录起始由o因子介导，其被分类为管家o70家族或主要变体o54。o因子招募RNA聚合酶到基因的上游启动子区域，并调节RNA聚合酶熔化和加载DNA到其活性位点以形成有能力的开放复合物的能力。o54启动子与营养消耗和应激反应相关，并且o54是独特的，因为它招募RNA聚合酶以形成稳定的封闭复合物。o54最初通过阻断DNA加载以及占据活性位点并位于RNA出口通道的顶部来抑制开放复合物的形成。因此，开放复合物的形成需要通过称为细菌增强子结合蛋白的进化枝6 AAA+（与多种细胞活性相关的ATP酶）蛋白的专门家族进行ATP水解。此外，o54必须经历一系列的构象变化，使启动子逃逸和过渡到进行性elongation.Using结合冷冻电子显微镜的生化方法，我们捕捉到多个AAA+从事中间状态，以阐明早期的机制，在开放复杂的形成过程中的DNA熔化。此外，我们已经确定了高分辨率的结构捕获的转录的初始阶段，深入了解如何o54在启动子escape.The工作在这里提出加深了我们的理解o54介导的转录起始，证明了保守的启动子逃逸在所有细菌的机制，并证明了一个独特的转录激活机制的AAA+蛋白质的一个专门的家庭。