SIGMA FACTOR IN DNA BINDING AND TRANSCRIPTION INITIATION

描述：原核生物RNA聚合酶都含有一个sigma亚基，该亚基 直接识别启动子内的特定序列元件，并通过 凭借这一点，将核心RNAP定向到启动子。几种不同的 西格玛因子由每个细菌物种编码，以启动转录 不同的基因组合。在大肠杆菌中，主要的西格玛因子西格玛70 允许大多数基因转录，而额外的替代西格玛 因子作用于启动一小部分启动子，通常是对 各种环境刺激或暗示。西格玛因子不仅在 RNAP的招募到了启动者，它还积极参与 启蒙过程，即。在启动子熔化和RNA链启动中。 这个项目的长期目标是提供一种分子理解 西格玛因子是如何起作用的，它的活动是如何被调控的。 Sigma因子都包含核心RNAP的结合域，它们 在其羧基末端含有两个特定的DNA结合域。这些 Sigma 7O中的DNA结合域识别以六聚体为中心的元件 分别位于起点的-10和-35处。在以前的工作中， 申请人已经证明，西格玛因子的DNA结合活性是 由于分子内相互作用而固有地被抑制，涉及氨基 蛋白质的末端部分，充当特定的DNA结合面具。 显然，当核心与Sigma结合时，DNA结合域被揭开 (这涉及到一个不同的领域)。 该提案的主要目标是：(A)了解两种DNA是如何 结合结构域在启动子识别过程中相互通信，(B) 以确定他们的活动是如何被西格玛的其他部分调节的 因素，以及(C)破译西格玛因素如何促进 封闭的促进剂络合物转变为开放的络合物和三元的络合物。突变型 主要群体和备选群体的西格玛因素的形式将 它们将在体内和体外的几个 方面：(I)DNA结合和干扰，(Ii)转录，以及(Iii) 特定的蛋白质-蛋白质相互作用。这些研究应该会增加我们的 基本转录机制的知识。

DESCRIPTION: Prokaryotic RNA polymerases all contain a sigma subunit that directly recognizes specific sequence elements within the promoter, and by virtue of this, directs the core RNAP to the promoter. Several different sigma factors are encoded by each bacterial species to turn on transcription of different sets of genes. In E. coli, the major sigma factor sigma 7O allows transcription of most genes, while additional alternative sigma factors act to turn on a small subset of promoters, often in response to various environmental stimuli or cues. The sigma factor not only mediates the recruitment of RNAP to the promoter, it also participates actively in the initiation process, ie. in promoter melting and RNA chain initiation. The long term goal of this project is to provide a molecular understanding of how sigma factor works and how its activity is regulated. The sigma factors all contain a binding domain for the core RNAP, and they contain two specific DNA binding domains in their carboxy termini. These DNA binding domains in sigma 7O recognize hexamer elements centered respectively at -10 and -35 from the start site. In previous work, the applicant has shown that the DNA binding activity of sigma factors is intrinsically inhibited due to intramolecular interactions, involving amino terminal parts of the protein that act as specific DNA binding masks. Obviously, the DNA binding domains are unmasked when the core binds to sigma (which involves a yet distinct domain). The major goals of the proposal are (a) to understand how the two DNA binding domains communicate with each other during promoter recognition, (b) to determine how their activities are modulated by other parts of the sigma factor, and (c) to decipher how the sigma factor promotes transition of the closed promoter complex to the open complex and the ternary complex. Mutant forms of sigma factors from both the major and the alternative groups will be generated, and they will be characterized in vivo and in vitro in several respects: (i) DNA binding and interference, (ii) transcription, and (iii) specific protein-protein interactions. These studies should increase our knowledge of the basic transcription mechanisms.