RNA POLYMERASE SIGMA SUBUNIT: STRUCTURE AND FUNCTION

现在已知RNA合成起始的调节是一个主要的 调节基因表达的机制， 细胞或生物体的能力。 许多疾病是由于 基因表达的错误调节。 因此， 理解转录的基本机制及其控制。 在定义转录因子方面已经取得了相当大的进展， 细菌中的机器，特别是RNA聚合酶的亚基。 的 已知σ亚基在确定选择性中是重要的， 体外和体内RNA链起始。 我们建议详细研究 E.大肠杆菌RNA聚合酶主要sigma 70， sigma 70与核心聚合酶和特异性启动子DNA的相互作用 序列和sigma家族的其他成员。 这样我们 我希望了解sigma 70是如何起作用的， RNA聚合酶结合和RNA链起始，以及细胞如何改变 它的转录模式通过使用其他sigma样因子。 1)我们将制备和表征单克隆抗体（MAb）试剂 需要研究E.大肠杆菌转录机制。 我们将准备MAB， 几个最近发现的sigma（sigmaE，sigmaF，sigmaK）。 我们有 开发了一种更有效的快速表位定位方法，并将使用它 来充分表征我们现在和未来的MAb。 我们将继续 改进我们免疫亲和纯化程序。 这些MAB是 对于我们进行体内亚基的定量是必要的， 亚基-亚基交联位点的定位，sigma 70的探测 结构和功能，以及快速纯化。 2)我们将继续研究sigma 70的结构和功能。 我们 将研究σ 70与核心聚合酶结合的位点。 我们将 确定每个MAb到sigma 70的转录过程中的哪个步骤 抑制。 我们已经开发出一种快速生产蛋白质的方法 片段，并将其用于表征σ 70的功能结构域。 我们将在2-D和3-D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 sigma家族成员与核心竞争。 我们将提纯毫克 纯化系统中核心聚合酶的已知σ的量 体外和体内。 我们将确定涉及的核心区域 与各种sigma的相互作用。

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.