Transcriptional Control by the Global Regulator Spx

全局调节器 Spx 的转录控制

• 批准号: 6926803
• 负责人: PETER ZUBER
• 金额: $ 35.37万
• 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/6926803
• 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

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响应的特征在于基因表达的全基因组变化，这些变化由对转录产生全局影响的因子协调。这些因子是致病菌中重要的毒力决定因子，因为它们控制细胞对宿主免疫系统施加的杀菌条件（例如氧化应激）的反应。来自枯草芽孢杆菌的蛋白质Spx在全基因组范围内响应于氧化应激而负性和正性地控制转录。Spx在低GC革兰氏阳性细菌中是保守的，具有类似于氧化还原因子如硫氧还蛋白中发现的活性位点的CXXC基序，并且由ATP依赖性蛋白酶C1 pXP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。该项目的目标是了解Spx如何发挥负和正转录控制，以及Spx浓度如何调节。将通过进行突变分析以鉴定alphaCTD上的激活因子和Spx相互作用表面，来检验Spx竞争alphaCTD上转录激活因子靶向位点的假设。将通过使用DNA-蛋白质交联和抑制子分析确定Spx-RNAP转录复合物的组成来研究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在CXXC基序中具有残基取代的突变体版本的激活和抑制活性。巯基特异性试剂和质谱法将用于鉴定活性形式Spx中Cys残基的推定修饰。氧化应激诱导的spx基因的转录控制将通过鉴定顺式作用和反式作用调节因子来研究。将通过确定C1 pXP的C1 pX亚基的锌指结构域是否是氧化还原依赖性调节的位点来研究Spx蛋白水解控制。将在体外测定氧化剂对Zn释放和C1 pXP活性的影响。