Elucidation of the regulation of Escherichia coli fis gene promoter by interactions between a module of RNA polymerase molecules and DNA architectural proteins

通过 RNA 聚合酶分子模块和 DNA 结构蛋白之间的相互作用阐明大肠杆菌 fis 基因启动子的调节

• 批准号: 49352938
• 负责人: Professor Dr. Georgi Muskhelishvili
• 金额: --
• 依托单位: Laboratoire de Microbiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2007
• 资助国家: 德国
• 起止时间: 2006-12-31 至 2009-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/49352938?language=en
• 关键词: Elucidation; regulation; Escherichia; coli; fis

Initiation of bacterial gene transcription is subject to complex regulation by concerted actions of DNA architectural transcription factors, protein cofactors of transcription, small effector molecules and the DNA topology. In addition, the promoter regions of certain pleiotropic genes demonstrate arrays of multiple RNA polymerase (RNAP) binding sites. The expression of the major chromatin protein FIS is regulated by several DNA architectural transcription factors, including CRP, IMF, H-NS and FIS itself. Previously we identified a module of RNAP binding sites in the regulatory region of Escherichia colt fis operon. These RNAP binding sites demonstrate strong promoter activities when cloned separately on plasmid constructs, but their function in the chromosomal context remains controversial - it is unclear whether the identified polymerase binding sites serve as true promoters, or have a purely regulatory function. Especially intriguing is the role of a overlapping module of fisP2 and a divergent promoter (divP/fisP2 module) identified upstream of the fisP1 site. We observed that an up mutation of the d/VP -10 hexamer increases the d/V transcription both in in vivo and in vitro. We also found that this mutation not only affects the binding of polymerase at the divP/fisP2 module but also at the fisP1 site, suggesting that upon occupation of these two sites the RNAP molecules interact with each other. In keeping with this notion, by using atomic force microscopy we observed that simultaneous binding of divP/fisP2 module and fisP1 sites stabilizes a unique complex containing at least two polymerase molecules presumably forming a RNAP dimer. Thus, our preliminary data strongly suggest a novel mechanism of transcriptional control involving direct interactions between RNAP molecules. In this project we intend to use the model system of divP/fisP2 and fisP1 promoters to investigate this novel regulation mechanism. We are keen to elucidate the relationships between the polymerase dimer formation at the fis promoter and the regulatory effects on fis transcription exerted by cAMP-CRP, IHF, H-NS and FIS itself. We believe this study will provide new insights into the diversity of transcriptional control mechanisms.

细菌基因转录的启动受到DNA结构转录因子、转录蛋白辅因子、小效应分子和DNA拓扑结构共同作用的复杂调控。此外，某些多效性基因的启动子区域显示出多个RNA聚合酶（RNAP）结合位点的阵列。主要染色质蛋白FIS的表达受几种DNA结构转录因子的调控，包括CRP、IMF、H-NS和FIS本身。在此之前，我们在大肠杆菌的操纵子调控区域发现了一个RNAP结合位点模块。这些RNAP结合位点在质粒上单独克隆时显示出很强的启动子活性，但它们在染色体上的功能仍然存在争议——目前尚不清楚所鉴定的聚合酶结合位点是作为真正的启动子，还是具有纯粹的调节功能。特别有趣的是fisP2的重叠模块和fisP1位点上游鉴定的发散启动子（divP/fisP2模块）的作用。我们观察到d/VP -10六聚体的up突变增加了体内和体外的d/V转录。我们还发现这种突变不仅影响聚合酶在divP/fisP2模块上的结合，还影响fisP1位点的结合，这表明当RNAP分子占据这两个位点时，它们会相互作用。根据这一观点，通过使用原子力显微镜，我们观察到divP/fisP2模块和fisP1位点的同时结合稳定了一个独特的复合物，该复合物含有至少两个聚合酶分子，可能形成RNAP二聚体。因此，我们的初步数据强烈暗示了一种涉及RNAP分子之间直接相互作用的转录控制新机制。在本项目中，我们打算使用divP/fisP2和fisP1启动子的模型系统来研究这种新的调控机制。我们渴望阐明fis启动子聚合酶二聚体的形成与cAMP-CRP、IHF、H-NS和fis本身对fis转录的调控作用之间的关系。我们相信这项研究将为转录控制机制的多样性提供新的见解。