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

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基因表达的全基因组变化，这些变化是由对转录产生全局影响的因素精心策划的。这些因子是致病菌重要的毒力决定因素，因为它们控制细胞对杀菌条件的反应，例如宿主免疫系统施加的氧化应激。来自枯草芽孢杆菌的一种蛋白质 Spx 可以在全基因组范围内负向和正向控制转录，以响应氧化应激。 Spx 在低 GC 革兰氏阳性细菌中是保守的，具有 CXXC 基序，类似于硫氧还蛋白等氧化还原因子中的活性位点，并由 ATP 依赖性蛋白酶 C1pXP 进行蛋白水解控制。在氧化环境中，Spx 通过与 RNA 聚合酶 (RNAP) α 亚基 (alphaCTD) 的 C 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 如何发挥负转录控制和正转录控制，以及 Spx 浓度如何调节。 Spx 竞争 alphaCTD 上转录激活剂靶向位点的假设将通过进行突变分析来测试，以识别 alphaCTD 上的激活剂和 Spx 相互作用表面。 Spx 依赖性阳性对照将通过使用 DNA-蛋白质交联和抑制分析确定 Spx-RNAP 转录复合物的组成来研究。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CXXC基序中具有残基取代的突变体版本的激活和抑制活性。硫醇特异性试剂和质谱将用于鉴定 Spx 活性形式中 Cys 残基的假定修饰。将通过鉴定顺式作用和反式作用调节因子来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 指结构域是否是氧化还原依赖性调节位点来研究 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测定。