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

细菌基因转录的起始受到DNA结构转录因子、转录的蛋白质辅因子、小效应分子和DNA拓扑结构的协同作用的复杂调控。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。我们在大肠杆菌操纵子调控区发现了RNAP结合位点模块。当在质粒构建体上单独克隆时，这些RNAP结合位点表现出强的启动子活性，但它们在染色体背景下的功能仍然存在争议-尚不清楚所鉴定的聚合酶结合位点是否充当真正的启动子，或具有纯粹的调节功能。特别有趣的是一个重叠模块的fisP 2和一个分歧的启动子（divP/fisP 2模块）的fisP 1网站的上游确定的作用。我们观察到d/VP-10六聚体的向上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/fisP 2模块的结合，但也在fisP 1网站，这表明在占领这两个网站的RNAP分子相互作用。与此概念一致，通过使用原子力显微镜，我们观察到，同时结合divP/fisP 2模块和fisP 1网站稳定了一个独特的复合物，含有至少两个聚合酶分子，大概形成RNAP二聚体。因此，我们的初步数据强烈建议一种新的转录控制机制，涉及RNAP分子之间的直接相互作用。在本项目中，我们打算使用divP/fisP 2和fisP 1启动子的模型系统来研究这种新的调控机制。我们热衷于阐明fis启动子处的聚合酶二聚体形成与cAMP-CRP、IHF、H-NS和FIS本身对fis转录的调节作用之间的关系。我们相信这项研究将为转录调控机制的多样性提供新的见解。

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.