RNAP ALPHA--DOMAIN ORGANIZATION, STRUCTURE & DNA BINDING

RNAP ALPHA--域组织、结构

• 批准号: 2190113
• 负责人: RICHARD H. EBRIGHT
• 金额: $ 25.53万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2190113
• 关键词: DNA binding protein; DNA directed RNA polymerase; X ray crystallography; bacteriophage M13; circular dichroism; crosslink; gene expression; genetic promoter element; nuclear magnetic resonance spectroscopy; protein structure function; stoichiometry; transcription factor

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.

大肠杆菌RNA聚合酶具有亚基组成 （alpha）2 β β sigma。 RNA聚合酶α亚基执行三个 重要功能（i）α作为RNA聚合酶的起始物 组装，（ii）α参与启动子识别的直接 序列特异性蛋白质-DNA相互作用，和（iii）α是靶 对于大量的转录激活蛋白，包括分解代谢物， 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 长期目标：（一）确定 RNA聚合酶的结构，（ii）理解启动子识别， (iii)理解转录起始，以及（iv）理解 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alpha的域组织。 实验 将A分解成抗蛋白酶的蛋白片段 （“结构域”），以确定N-末端和C-末端的边界。 α结构域，并分析寡聚化性质，组装 特性和α结构域的DNA结合特性。 具体目标2：分析α和α结构域的结构。 实验提出准备克数量的α和α 结构域，通过CD测定二级结构组成 光谱法，并通过多维分析确定三级结构 核磁共振光谱学和X射线晶体学。 具体目标3：通过α分析DNA结合。 实验 建议开发用于α-DNA的术前和定量分析 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;用于定量DNA结合 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 5-溴尿嘧啶介导的光交联，位点特异性脱甲基-脱碱基- 位点介导的交联和丙氨酸扫描诱变;以及 分析α DNA相互作用在转录起始中的作用。 获得的结果将直接关系到理解 原核基因表达的调控。 由于真核RNA 聚合酶亚基与大肠杆菌具有序列和机制上的同源性。 大肠杆菌RNA聚合酶亚基，包括a，结果得到 也可能与理解真核基因的调控有关 表情