STRUCTURAL STUDIES OF BACILLUS SUBTILIS SIGMA FACTORS

枯草芽孢杆菌 Sigma 因子的结构研究

• 批准号: 2701559
• 负责人: John D Helmann
• 金额: $ 16.64万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2701559
• 关键词: Bacillus subtilis; DNA directed RNA polymerase; DNA footprinting; DNA replication; Escherichia coli; SDS polyacrylamide gel electrophoresis; bacterial genetics; bacterial proteins; crosslink; deoxyribonuclease I; enzyme activity; enzyme mechanism; flagellin; gel mobility shift assay; gene expression; gene mutation; genetic promoter element; genetic regulatory element; photochemistry; protein reconstitution; protein sequence; protein structure function; regulatory gene; site directed mutagenesis; transcription factor

Transcription, catalyzed by RNA polymerase (RNAP), is the copying of genetic information from its DNA repository into functional RNA molecules. RNAP is a complex protein containing several subunits with distinct functions. In recent years it has become clear that the structure of RNAP has been evolutionarily conserved from bacteria to man. Our work focuses on RNAP from Bacillus subtilis, a gram positive soil microbe. Uke most bacterial RNAPs, this enzyme contains a multi-subunit core enzyme (beta beta'alpha2) associated with one of several specificity factors known as sigma. The sigma subunit determines where transcription initiates by allowing RNAP to recognize specific promoter sites and form a transcriptionally competent open (strand-separated) complex. Most transcription during growth requires the primary sigma, sigmaA. However, this sigma subunit is replaced by alternative sigma factors to allow the expression of specialized sets of genes. In addition, B. subtilis RNAP contains delta, a nonessential protein which influences enzyme activity and promoter recognition. Ultimately, we hope to understand the molecular determinants of promoter recognition, the structure of and its subunits, and transcription initiation and its regulation. The biochemical activities of sigma factor during transcription initiation will be studied by analysis of RNAP promoter complexes using biochemical and molecular genetic techniques. These experiments will address the hypothesis that a conserved region of sigma factors, region 2.3, binds to the single-stranded DNA of the transcription bubble. To test this idea, RNAP-promoter complexes will be analyzed by photocrosslinking. The role of sigma in determining the detailed pathway of transcription initiation will be investigated by comparative studies of three RNAP holoenzymes; E- sigmaA, E-sigmaD, and E-sigmaX. Finally, the role of delta in determining promoter selectivity will be studied both in vitro and in vivo. These mechanistic and structural analyses of RNAP m Bacillus subtilis will: (i) provide a necessary context for understanding transcriptional regulation in this and related organisms; (ii) provide a useful test for paradigms developed largely from study of E. coli RNAP; and (iii) allow insights into the roles of structurally related subunits of eukaryotic RNAP.

转录是由 RNA 聚合酶 (RNAP) 催化的，是复制 遗传信息从 DNA 库转化为功能性 RNA 分子。 RNAP 是一种复杂的蛋白质，包含多个具有不同功能的亚基 功能。近年来，RNAP的结构已逐渐明确。 从细菌到人类，在进化上一直是保守的。我们的工作重点 枯草芽孢杆菌（一种革兰氏阳性土壤微生物）的 RNAP。最喜欢 细菌 RNAP，该酶含有多亚基核心酶（β beta'alpha2）与称为 西格玛。西格玛亚基决定转录起始位置 允许RNAP识别特定的启动子位点并形成 具有转录能力的开放（链分离）复合物。最多 生长过程中的转录需要初级西格玛，sigmaA。然而， 该西格玛亚基被替代西格玛因子取代，以允许 特殊基因组的表达。此外，枯草芽孢杆菌 RNAP 含有 Delta，一种影响酶活性的非必需蛋白质 和发起人的认可。最终，我们希望了解分子 启动子识别的决定因素，及其亚基的结构， 以及转录起始及其调控。 转录起始过程中σ因子的生化活性 将通过使用生化分析 RNAP 启动子复合物来研究 和分子遗传学技术。这些实验将解决 假设 sigma 因子的保守区域（区域 2.3）结合 转录泡的单链DNA。为了测试这个想法， RNAP-启动子复合物将通过光交联进行分析。的作用 西格玛确定转录起始的详细途径将 通过三种 RNAP 全酶的比较研究进行研究；电子- sigmaA、E-sigmaD 和 E-sigmaX。最后，Delta 在决定中的作用 将在体外和体内研究启动子选择性。这些 RNAP m 枯草芽孢杆菌的机制和结构分析将： (i) 为理解转录调控提供必要的背景 在该生物体和相关生物体中； (ii) 为范式提供有用的测试 主要是从大肠杆菌 RNAP 的研究中发展而来； (iii) 允许洞察 研究真核 RNAP 结构相关亚基的作用。