E COLI RNA POLYMERASE SUBUNIT--STRUCTURE AND FUNCTION

我们的目标是了解转录的机制和它的 监管。确定RNA的结构/功能关系 聚合酶，负责rna合成的酶，是必不可少的一步。 朝着这个目标前进。这最好是通过高度特征化的 E.ColiRNA聚合酶。由于其庞大的规模和复杂性，这 将需要生化和生物物理方法的组合来 鉴定和鉴定RNAP亚单位的结构域，X射线 用结晶学方法确定RNA聚合酶的高分辨结构 亚单位结构域和电子显微镜来确定完整的结构 装配。 我们自己的工作和其他人的工作的许多证据表明 这种RNA聚合酶在其结构上是模块化的。大肠杆菌核糖核糖核酸 聚合酶由单独的多肽亚基组成，具有 化学计量比α2betabeta‘sigmna。然而，各个亚基， 似乎是由独立的子域构成的，这些子域具有部分 他们自己的功能。这项提案的目标是确定和 描述组成RNA聚合酶亚单位的亚域的特征。 更具体地说，就每个亚单位而言： 1)β亚基。我们已经开始阐明模数 用一种新的体外组装和功能测试方法构建β 使用贝塔片段的RNA聚合酶。使用同样的方法，我们 将进一步定义测试亚单位模块。详细的功能研究 将介绍每个模块在以下方面的角色：1)与阿尔法的互动 RNA聚合酶组装，2)底物核苷酸结合，以及3) 转录结合和修改。蛋白质水解物的详细分析 带有beta的站点将被用来描述beta亚基的结构特征 模块。 2)β‘亚基。我们将使用相同的方法来剖析测试版； 子单元组成其模块化组件。功能研究将解决 β‘模块在RNAP中与α2β相互作用中的作用 集合。原生生物裂解研究将用于结构表征 Beta‘亚单位模块。 3)Sigma70亚基。我们已经进行了研究，以定义和结构 用蛋白水解法鉴定Sigma70结构域。我们将确定X- 含有守恒的子区晶体的射线晶体结构 区域2，它与-10启动子的共同序列相互作用。 功能研究将解决sigma70结构域在以下方面的作用：1) 与核心RNAP的结合和特定启动子的结合和/或熔化 元素。 4)α亚基。阿尔法亚单位的结构域一直是 被其他人阐明的。我们会进行详细的结构研究。 用X射线结晶学的方法研究了α亚基结构域。

Our goal is to understand the mechanism of transcription and its regulation. Determining the structure/function relationship of RNA polymerase, the enzyme responsible for RNA synthesis, is an essential step towards this goal. This is best accomplished with the highly characterized E. coli RNA polymerase. Because of the large size and complexity, this will require a combination of biochemical and biophysical methods to identify and characterize domains of the RNAP subunits, X-ray crystallography to determine high-resolution structures of RNA polymerase subunit domains, and electron microscopy to determine structures of intact assemblies. Many lines of evidence from our own work an the work of others indicate that RNA polymerase is modular in its architecture. The E. coli RNA polymerase is comprised of individual polypeptide subunits with stoichiometry alpha2betabeta'sigmna. The individual subunits, however, seem to be constructed of independent subdomains that have partial functions of their own. The goal of this proposal is to identify and characterize the subdomains that comprise the RNA polymerase subunits. More specifically, in terms of each subunit: 1) beta Subunit. We have already begun to elucidate the modular architecture of beta with a novel assay of in vitro assembly and function of RNA polymerase using fragments of beta. Using the same approach, we will further define the beta subunit modules. Detailed functional studies will address the roles of each module in; 1) the interaction with alpha in RNA polymerase assembly, 2) substrate nucleotide binding, and 3) transcript binding and modification. Detailed analysis of proteolytic sites with beta will be used to structurally characterize the beta subunit modules. 2) beta' Subunit. We will use the same approaches to dissect the beta; subunit into its modular components. Functional studies will address the roles of the beta' modules in the interaction with alpha2beta in RNAP assembly. Protoeolytic studies will be used to structurally characterize the beta' subunit modules. 3) sigma70 Subunit. We have undertaken studies to define and structurally characterize sigma70 domains using proteolysis. We will determine the X- ray crystal structure from crystals of a sub-domain containing conserved region 2, which interacts with the -10 promoter consensus sequence. Functional studies will address the roles of sigma70 domains in; 1) binding to core RNAP and binding and/or melting of specific promoter elements. 4) alpha Subunit. The domain architecture of the alpha subunit has been elucidated by others. We will undertake detailed structural studies of alpha subunit domains using the method of X-ray crystallography.