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，由RNA聚合酶（RNAP）催化，是复制 从其DNA存储库中的遗传信息到功能性RNA分子。 RNAP是一种复杂的蛋白质，包含几个具有不同的亚基 功能。近年来，RNAP的结构很明显 从细菌到人的进化保守了。我们的工作重点 在枯草芽孢杆菌的RNAP上，革兰氏阳性土壤微生物。最大 细菌rnap，该酶含有多种亚基核心酶（β beta'alpha2）与称为的几个特异性因素之一相关 西格玛。 Sigma亚基确定转录在何处通过 允许RNAP识别特定的启动子站点并形成A 具有转录能力的开放式（链分离）复合物。最多 生长过程中的转录需要初级的Sigma，Sigmaa。然而， 该Sigma亚基被替代的Sigma因子取代 专门基因集的表达。此外，枯草芽孢杆菌rnap 包含三角洲，这是一种不必要的蛋白质，影响酶活性 和发起人的认可。最终，我们希望理解分子 启动子识别，结构及其亚基的决定因素， 和转录启动及其调节。 转录启动过程中Sigma因子的生化活性 将通过使用生化的RNAP启动子复合物分析来研究 和分子遗传技术。这些实验将解决 假设Sigma因子的保守区域2.3与 转录气泡的单链DNA。为了测试这个想法， RNAP启动器复合物将通过Photocroslinking进行分析。的作用 Sigma确定详细的转录启动途径将 通过对三个RNAP全酶的比较研究来研究； e- Sigmaa，E-Sigmad和E-Sigmax。最后，三角洲在确定 启动子选择性将在体外和体内研究。这些 RNAP M枯草芽孢杆菌的机械和结构分析将：（i） 提供了理解转录监管的必要背景 在这个和相关的生物中； （ii）为范例提供了有用的测试 主要是由大肠杆菌RNAP的研究发展的； （iii）允许见解 进入真核RNAP的结构相关亚基的作用。