RNAP ALPHA--DOMAIN ORGANIZATION, STRUCTURE & DNA BINDING

大肠杆菌RNA聚合酶具有亚基组成 (Alpha)2BetaBetasigma。RNA聚合酶α亚基执行三个 重要功能(I)α作为RNA聚合酶的启动子 组装，(Ii)α通过直接参与启动子识别 序列特异的蛋白质-DNA相互作用，以及(Iii)α是靶标 对于一大套转录激活蛋白，包括分解代谢 基因激活蛋白(CAP)。 我们关于RNA聚合酶α亚基的研究项目针对四个 远景目标：(一)三维目标的确定 RNA聚合酶的结构，(Ii)理解启动子识别， (Iii)了解转录启动，以及(Iv)了解 转录激活。我们的研究项目有三个具体目标： 具体目标1：分析阿尔法的领域组织。实验 被提议将a分解成抗蛋白酶的蛋白质片段 (“域”)，以确定N-端和C-端的边界 Alpha结构域，并分析齐聚性能，组装 特性和α结构域的DNA结合特性。 具体目标2：分析阿尔法和阿尔法结构域的结构。 建议进行实验以制备克量的α和α 结构域，用CD确定二级结构组成 光谱学，并用多维方法确定三级结构 核磁共振谱和X射线结晶学。 具体目标3：分析α与DNA的结合。实验是 建议开展术前α-DNA定量检测 相互作用；确定“最佳”DNA位点序列和最小 α-DNA相互作用的DNA位点长度；以量化DNA结合 4的化学计量、DNA结合亲和力和DNA结合特异性 α-DNA相互作用；通过位点特异性鉴定DNA结合残基 5-溴沙拉星介导的光交联，定点的醛-碱- 位点介导的交联剂和丙氨酸扫描诱变； 分析αDNA相互作用在转录启动中的作用。 所获得的结果将直接关系到对 原核生物基因表达的调控。由于真核RNA 聚合酶亚基与E. ColiRNA聚合酶亚基，包括a，结果待获得 也可能与理解真核基因的调控有关。 表情。

Escherichia coli RNA polymerase has subunit composition (alpha)2BetaBetasigma. RNA polymerase alpha subunit carries out three important functions (i) alpha serves as the initiator for RNA polymerase assembly, (ii) alpha participates in promoter recognition by direct sequence-specific protein-DNA interaction, and (iii) alpha is the target for a large set of transcription activator proteins, including catabolite gene activator protein (CAP). Our research project on RNA polymerase alpha subunit is directed at four long-range objectives: (i) determination of the three-dimensional structure of RNA polymerase, (ii) understanding promoter recognition, (iii) understanding transcription initiation, and (iv) understanding transcription activation. Our research project has three specific aims: Specific Aim 1: Analysis of domain organization of alpha. Experiments are proposed to dissect a into protease-resistant protein fragments ("domains), to determine the N-terminal and C-terminal boundaries of alpha domains, and to analyze the oligomerization properties, assembly properties, and DNA binding properties of alpha domains. Specific Aim 2: Analysis of structure of alpha and alpha domains. Experiments are proposed to prepare gram quantities of alpha and alpha domains, to determine secondary structure compositions by CD spectroscopy, and to determine tertiary structures by multi-dimensional NMR spectroscopy and x-ray crystallography. Specific Aim 3: Analysis of DNA binding by alpha. Experiments are proposed to develop preoperative and quantitative assays for alpha-DNA interaction; to determine the "optimal" DNA-site sequence and minimum DNA-site length for alpha-DNA interaction; to quantify the DNA binding stoichiometry, DNA binding affinity, and DNA binding specificity of 4 alpha-DNA interaction; to identify DNA binding residues by site-specific 5-bromouracil-mediated photocrosslinking, site-specific aldehydic-abasic- site-mediated crosslinking, and alanine-scanning mutagenesis; and to analyze the role of alphaDNA interaction in transcription initiation. The results to be obtained will be directly relevant to understanding regulation of prokaryotic gene expression. Since eukaryotic RNA polymerase subunits share sequence and mechanistic homologies with E. coli RNA polymerase subunits, including a, the results to be obtained also may be relevant to understanding regulation of eukaryotic gene expression.