SIGMA FACTOR IN DNA BINDING AND TRANSCRIPTION INITIATION

描述：前体RNA聚合酶都含有一个σ亚基， 直接识别启动子内的特定序列元件， 因此，将核心RNAP导向启动子。 几种不同 每个细菌物种编码σ因子以开启转录 不同的基因组。 在大肠大肠杆菌，主要的σ因子σ 7 O 允许大多数基因的转录，而额外的替代西格玛 因子作用于启动子的一小部分，通常是响应于 各种环境刺激或线索。 sigma因子不仅介导 RNAP的招募发起人，它还积极参与 启动过程，即。 启动子解链和RNA链起始。 这个项目的长期目标是提供一个分子理解 sigma因子是如何工作的以及它的活动是如何被调节的。 sigma因子都含有核心RNAP的结合域，并且它们 在其羧基末端含有两个特异性DNA结合结构域。 这些 sigma 7 O中的DNA结合结构域识别六聚体元件 分别在距起始位置-10和-35处。 在以前的工作中， 申请人已经表明，σ因子的DNA结合活性是 由于分子内相互作用而固有抑制，涉及氨基 作为特异性DNA结合掩膜的蛋白质末端部分。 显然，当核心与σ结合时， （涉及一个不同的领域）。 该提案的主要目标是（a）了解两个DNA如何 结合结构域在启动子识别过程中相互通讯，（B） 以确定他们的活动是如何被sigma的其他部分调节的 因子，以及（c）破译sigma因子如何促进 封闭启动子复合物与开放复合物和三元复合物的比例。 突变体 主要和备选组的sigma因子形式将 产生，它们将在体内和体外在几个 （i）DNA结合和干扰，（ii）转录，和（iii） 特异性蛋白质间相互作用。 这些研究应该增加我们的 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.