E COLI RNA POLYMERASE SUBUNIT--STRUCTURE AND FUNCTION

我们的目标是了解转录的机制及其 调控 确定RNA的结构/功能关系 负责RNA合成的聚合酶是一个重要步骤， 朝着这个目标前进。 这是最好的完成与高度表征 e. coli RNA聚合酶。 由于其庞大的规模和复杂性， 将需要生物化学和生物物理方法的结合， 识别和表征RNAP亚基的结构域，X射线 晶体学来确定RNA聚合酶的高分辨率结构 亚基结构域和电子显微镜，以确定完整的 组件. 从我们自己的工作和其他人的工作中得到的许多证据表明， RNA聚合酶的结构是模块化的。 急诊杆菌rna 聚合酶由单独的多肽亚基组成， 化学计量α 2 β 2 σ 然而，各个子单位， 似乎是由独立的子域构成的， 自己的功能。 本提案的目标是确定和 表征包含RNA聚合酶亚基的亚结构域。 更具体地说，就每个子单元而言： 1)贝塔亚单位。 我们已经开始阐明模块化 β的结构与体外组装和功能的新测定 RNA聚合酶的基因片段。 使用相同的方法，我们 将进一步定义β亚基模块。 详细的功能研究 将说明中每个模块的作用; 1）与中alpha的交互 RNA聚合酶组装，2）底物核苷酸结合，和3） 转录本结合和修饰。 蛋白水解的详细分析 具有β的位点将用于结构表征β亚基 模块。 2)贝塔亚单位。 我们将使用相同的方法来剖析测试版; 子单元到其模块化组件。 功能研究将解决 RNAP中β '模块在与α 2 β相互作用中的作用 组装件. 将使用原核溶解研究来表征 β亚基模块。 3)sigma70亚单位。 我们已经进行了研究， 使用蛋白水解表征σ 70结构域。 我们将确定X- 射线晶体结构从晶体的一个子域含有保守的 区域2，其与-10启动子共有序列相互作用。 功能研究将解决sigma70结构域在以下方面的作用：1） 与核心RNAP的结合以及特异性启动子的结合和/或解链 元素 4)阿尔法亚单位 α亚基的结构域结构已经被 由其他人解释。 我们会进行详细的结构研究， α亚基结构域的X射线晶体学方法。

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.