Transcriptional Control by the Global Regulator Spx

全局调节器 Spx 的转录控制

• 批准号: 7025041
• 负责人: PETER ZUBER
• 金额: $ 35.29万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7025041
• 关键词: Bacillus subtilis; DNA directed RNA polymerase; bacterial genetics; bacterial proteins; calorimetry; disulfide bond; electron transport; enzyme activity; gel mobility shift assay; gene mutation; genetic promoter element; genetic regulation; genetic regulatory element; mass spectrometry; operon; oxidative stress; protein structure function; serine proteinases; thiols; transcription factor; western blottings; yeast two hybrid system; Bacillus subtilis; DNA directed RNA polymerase; bacterial genetics; bacterial proteins; calorimetry; disulfide bond; electron transport; enzyme activity; gel mobility shift assay; gene mutation; genetic promoter element; genetic regulation; genetic regulatory element; mass spectrometry; operon; oxidative stress; protein structure function; serine proteinases; thiols; transcription factor; western blottings; yeast two hybrid system

DESCRIPTION (provided by applicant): A microorganism's response to a harsh environment is characterized by genome-wide changes in gene expression orchestrated by factors that exert a global influence over transcription. These factors are important virulence determinants in pathogenic bacteria because they control the cell's response to the bacteriocidal conditions, such as oxidative stress, imposed by the host's immune systems. A protein from Bacillus subtilis, Spx, both negatively and positively controls transcription over a genome-wide scale in response to oxidative stress. Spx is conserved among low GC Gram-positive bacteria, bears a CXXC motif resembling an active site found in redox factors such as thioredoxin, and is controlled proteolytically by the ATP-dependent protease, C1pXP. In an oxidative environment, Spx affects transcription by interacting with the C-terminal domain of the RNA polymerase (RNAP) alpha subunit (alphaCTD), but it does not itself bind to DNA. The goal of the proposed project is to understand how Spx exerts both negative and positive transcriptional control, and how Spx concentration is regulated. The hypothesis that Spx competes for sites on alphaCTD that are targeted by transcription activators will be tested by conducting mutational analysis to identify activator and Spx interaction surfaces on alphaCTD. Spx-dependent positive control will be studied by determining the composition of the Spx-RNAP transcription complex using DNA-protein crosslinking and suppressor analysis. To further understand the redox control of Spx, the activation and repression activities of mutant versions having residue substitutions in the CXXC motif will be analyzed by in vitro transcription experiments. Thiol-specific reagents and mass spectrometry will be used to identify putative modifications of the Cys residues in the active form of Spx. The transcriptional control of the oxidative stress-induced spx gene will be studied by identifying both cis-acting and trans-acting regulatory factors. Spx proteolytic control will be studied by determining if the Zn-finger domain of the C1pX subunit of C1pXP is the site of redoxdependent regulation. The effects of oxidants on Zn release and C1pXP activity will be determined in vitro.

描述（由申请人提供）：微生物对恶劣环境的反应的特征是全基因表达的基因表达的变化由对全球影响转录产生全球影响的因素策划。这些因素是致病细菌中重要的毒力决定因素，因为它们控制了细胞对杀菌条件的反应，例如宿主的免疫系统施加的氧化应激。枯草芽孢杆菌，SPX的蛋白质对氧化应激的响应在全基因组尺度上进行负和正面控制。 SPX是在低GC革兰氏阳性细菌中保守的，具有类似于氧化还原因子（例如硫氧还蛋白）的活性位点的CXXC基序，并由ATP依赖性蛋白酶C1PXP通过蛋白水解控制。在氧化环境中，SPX通过与RNA聚合酶（RNAP）α亚基（AlphACTD）的C末端结构域相互作用来影响转录，但它本身不与DNA结合。拟议项目的目的是了解SPX如何施加负转录和阳性转录控制，以及如何调节SPX浓度。通过进行突变分析以识别AlphACTD上的激活剂和SPX相互作用表面，将测试SPX竞争AlphACTD竞争AlphACTD的位点的假设。通过使用DNA-蛋白交联和抑制器分析来确定SPX-RNAP转录复合物的组成，将研究SPX依赖性阳性对照。为了进一步了解SPX的氧化还原控制，将通过体外转录实验分析具有残留替代的突变版本的激活和抑制活性。硫醇特异性试剂和质谱法将用于鉴定SPX活性形式的CYS残基的假定修饰。通过识别顺式作用和跨作用调节因子，将研究氧化应激诱导的SPX基因的转录控制。通过确定C1PXP的C1PX亚基的Zn手指域是否是依赖氧化还原的调节的位点，将研究SPX蛋白水解控制。氧化剂对Zn释放和C1PXP活性的影响将在体外确定。