RNA POLYMERASE SIGMA SUBUNIT: STRUCTURE AND FUNCTION

现在已知对RNA合成的启动的调节是一个主要的 调节基因表达的机制，从而调节酶 细胞或有机体的能力。许多疾病都是由 对基因表达的错误调控。因此，重要的是， 了解转录及其控制的基本机制。 在定义转录本方面已经取得了相当大的进展 细菌中的机械，特别是RNA聚合酶的亚单位。这个 已知Sigma亚基在决定药物选择性中起重要作用 体外和体内核糖核酸链的起始。我们建议详细研究 大肠杆菌RNA聚合酶主要西格玛(Sigma70)的结构， Sigma70与核心聚合酶和特异性启动子DNA的相互作用 序列，以及西格玛家族的其他成员。通过这种方式，我们 希望了解sigma70是如何发挥作用来决定 RNA聚合酶结合和RNA链启动，以及细胞如何改变 它的转录模式通过使用其他西格玛样因子。 1)我们将制备并鉴定单抗试剂。 需要研究大肠杆菌转录机制。我们将准备单抗以 最近发现的几个sigma(sigmaE、sigmaF、sigmaK)。我们有 开发了一种更有效的快速表位映射方法，并将使用它 来充分描述我们现在和未来的单抗。我们将继续 改进我们的免疫亲和纯化程序。这些单抗是 对于我们在体内进行亚基的量化是必不可少的， 亚基-亚基交联点的定位，Sigma70的探测 结构和功能，以及在这项拨款中提出的快速纯化。 2)我们将继续研究Sigma70的结构和功能。我们 将研究Sigma70与核心聚合酶的结合部位。我们会 确定每个单抗到sigma70的转录过程中的哪一步 抑制。我们开发了一种快速生产蛋白质的方法。 片段，并将用它来表征sigma70的功能结构域。 我们将在二维和三维晶体结构测定方面进行合作。 3)我们将研究全酶，特别是影响因素 以西格玛为核心的家族成员竞争。我们会提纯毫克 已知的核心聚合酶在纯化系统中的Sigma量 体外和体内。我们将确定核心上涉及的区域 与各种西格玛的互动。

Regulation of the initiation of RNA synthesis is now known to be a major mechanism for regulating gene expression and thus the enzymatic capabilities of a cell or organism. Many disorders are the result of faulty regulation of gene expression. It is important, therefore, to understand the basic mechanism of transcription and its control. Considerable progress has been made in defining the transcriptional machinery in bacteria, in particular the subunits of RNA polymerase. The sigma subunit is known to be important in determining the selectivity of RNA chain initiation in vitro and in vivo. We propose to study in detail the structure of the E. coli RNA polymerase major sigma (sigma70), the interactions of sigma70 with the core polymerase and specific promoter DNA sequences, and the additional members of the sigma family. In this way we hope to understand how sigma70 functions to determine the selectivity of RNA polymerase binding and RNA chain initiation, and how the cell can alter its pattern of transcription by using other sigma-like factors. 1) We will prepare and characterize monoclonal antibody (MAb) reagents needed to study E. coli transcription machinery. We will prepare MAbs to several recently discovered sigmas (sigmaE, sigmaF, sigmaK). We have developed a more effective method for rapid epitope mapping and will use it to fully characterize our present and future MAbs. We will continue to improve our immunoaffinity purification procedures. These MAbs are essential for us to carry out the quantitation of subunits in vivo, localization of subunit-subunit crosslink sites, probing of sigma70 structure and function, and rapid purification proposed in this grant. 2) We will continue our study of the structure and function of sigma70. We will study the site of binding of sigma70 to core polymerase. We will determine what step in the transcription process each MAb to sigma70 is inhibiting. We have developed a rapid method for producing protein fragments and will use it to characterize functional domains of sigma70. We will collaborate on 2-D and 3-D crystal structure determination. 3) We will study holoenzymes, particularly the factors affecting competition of sigma-family members with core. We will purify milligram amounts of the known sigmas for core polymerase both in purified systems in vitro and in vivo. We will determine the regions on core involved in interactions with various sigmas.