Transcriptional Control by the Global Regulator Spx

描述（由申请人提供）：微生物对恶劣环境的反应以基因表达的全基因组变化为特征，这些变化是由对转录施加全球影响的因素协调的。这些因素在致病菌中是重要的毒力决定因素，因为它们控制着细胞对宿主免疫系统施加的杀菌条件（如氧化应激）的反应。枯草芽孢杆菌（Bacillus subtilis, Spx）中的一种蛋白质，在氧化应激反应中，在全基因组范围内负性和正性控制转录。Spx在低GC革兰氏阳性细菌中保守，带有CXXC基元，类似于氧化还原因子（如硫氧还蛋白）中的活性位点，并受atp依赖性蛋白酶C1pXP的蛋白水解控制。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如何发挥负转录和正转录控制作用，以及Spx浓度如何受到调节。Spx竞争转录激活子靶向的alphaCTD上的位点的假设将通过进行突变分析来验证，以确定alphaCTD上的激活子和Spx相互作用表面。通过使用dna -蛋白交联和抑制因子分析来确定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，我们将通过体外转录实验分析CXXC基序中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法将用于鉴定活性形式Spx中Cys残基的假定修饰。氧化应激诱导的spx基因的转录控制将通过确定顺式作用和反式作用的调控因子来研究。通过确定C1pXP的C1pX亚基的锌指结构域是否为氧化还原依赖性调控位点，将研究Spx蛋白水解控制。体外测定不同氧化剂对锌释放和C1pXP活性的影响。

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.