RNA POLYMERASE SIGMA SUBUNIT--STRUCTURE AND FUNCTION

我们建议详细研究大肠杆菌RNA的结构 聚合酶主要Sigma亚单位(Sigma70)，Sigma70与Sigma的相互作用 核心聚合酶亚基和西格玛家族的其他成员 以及它们如何竞争与核心的绑定。通过这种方式，我们希望 了解sigma70如何发挥作用来决定RNA的选择性 聚合酶结合和核糖核酸链启动，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。 具体来说，我们的目标是： A.研究Sigma-70的结构和功能。我们将测试我们的 假设sigma70形成一个“发夹”结构，并且必须打开或 部分展开以发挥作用。我们将结晶Sigma70，确定它的 3-D晶体结构，并与其他晶体结构进行了比较。我们会 利用突变和蛋白质片段探索功能结构域 一种在大肠杆菌中快速生产蛋白质和蛋白质片段的方法 Coli-S-30提取系统。我们将确定 转录过程中每个单抗都抑制了Sigma70的表达。我们会做得更多 精确地绘制和研究几个特别有趣的单抗。 B.确定Sigma中涉及的核心聚合酶亚单位的区域 互动。我们将使用化学交联和特定的单抗来 确定Sigma70与25个氨基酸短区的结合部位 从Sigma70到核心。我们还将确定核心上涉及的区域 与其他西格玛的互动。 研究西格玛家族成员对核心的竞争。我们将衡量 体内已知Sigma的水平作为生长条件的函数。我们 将研究这些不同的Sigma对核心聚合酶的竞争 无论是在体内还是在体外纯化系统中。我们将研究这一角色 影响西格玛竞争的反西格玛。 D.研究Sigma-S和Sigma-70与BolAp1的全酶相互作用 推动者。我们将确定含有全酶的相互作用 与bolAp1启动子的Sigma70或Sigma，并确定为什么 全酶在体外发挥作用，但只有E-Sigma-S在体内发挥作用 推动者。 我们建议继续研究西格玛的结构和功能 生物化学、蛋白质和物理化学的协同使用，单抗。 技术和分子遗传学。我们长期研究西格玛和 我们最近的进展使我们处于一个独特的地位，可以执行 提出的实验和回答许多重要的遗留问题 关于大肠杆菌Sigma的结构和功能。

We propose to study in detail the structure of the Escherichia coli RNA polymerase major sigma subunit(sigma70),the interaction of sigma70 with core polymerase subunits, and the additional members of the sigma family and how they compete for binding to core. 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. Specifically, our aims are: A. To Study the Structure and Function of Sigma-70. We will test our hypothesis that sigma70 forms a "hairpin" structure and must open or partially unfold to function. We will crystallize sigma70, determine its 3-D crystal structure, and compare its structure to other sigmas. We will use mutations and protein fragments to probe functional domains utilizing a rapid method for producing proteins and protein fragments in Escherichia coli S-30 extract systems. We will determine what step in the transcription process each MAb to sigma70 is inhibiting. We will more precisely epitope map and study several particularly interesting MAbs. B. To Identify Regions of Core Polymerase Subunits Involved in Sigma Interaction. We will use chemical crosslinking and specific MAbs to determine the site of binding of sigma70 and a short 25 amino acid region of sigma70 to core. We will also determine the regions on core involved in interactions with other sigmas. C. To Study Competition of Sigma-Family Members for Core. We will measure the in vivo levels of known sigmas as a function of growth conditions. We will study the competition of these various sigmas for core polymerase both in vivo and in purified in vitro systems. We will examine the role of anti-sigmas in affecting sigma competition. D. To Study Sigma-S and Sigma-70 Holoenzyme Interaction with the bolAp1 Promoter. We will determine the interactions of holoenzyme containing sigma70 or sigmaS with the bolAp1 promoter and determine why both holoenzymes function in vitro but only E-sigma-S functions in vivo at this promoter. We propose to continue to study sigma structure and function by a concerted use of biochemistry, protein and physical chemistry, MAb. technology, and molecular genetics. Our long experience studying sigma and our recent progress place us in a unique position to carry out the proposed experiments and to answer many important remaining questions about the structure and function of Escherichia coli sigmas.