Transcriptional Control by the Global Regulator Spx

描述(申请人提供)：微生物对恶劣环境的反应的特征是基因表达在基因组范围内的变化，这些变化是由对转录产生全球影响的因素协调的。这些因子是病原菌的重要毒力决定因素，因为它们控制细胞对宿主免疫系统施加的氧化应激等杀菌条件的反应。枯草芽孢杆菌的一种蛋白质Spx在全基因组范围内负向和正向地控制转录，以响应氧化应激。SPX在低GC革兰氏阳性细菌中保守，带有一个CXXC基序，类似于硫氧还蛋白等氧化还原因子中的活性部位，并受ATP依赖的蛋白酶C1pXP控制。在氧化环境中，Spx通过与RNA聚合酶(RNAP)α亚单位(AlphaCTD)的C末端区域相互作用来影响转录，但它本身不与DNA结合。拟议项目的目标是了解Spx如何同时发挥负转录和正转录调控作用，以及Spx浓度是如何调节的。Spx竞争αCTD上转录激活子靶点的假设将通过突变分析来确定AlphaCTD上激活子和Spx的相互作用表面。依赖于Spx的阳性对照将通过DNA-蛋白质交联和抑制分析确定Spx-RNAP转录复合体的组成来研究。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来分析CXXC基序中含有残基替换的突变版本的激活和抑制活性。硫醇特异性试剂和质谱学将被用来鉴定Spx活性形式中Cys残基的假定修饰。氧化应激诱导的SPX基因的转录调控将通过识别顺式作用和反式作用的调节因子来研究。通过确定C1pXP的C1pX亚单位的锌指结构域是否是氧化还原依赖的调节部位，将研究SPX的蛋白降解控制。氧化剂对锌释放和C1pXP活性的影响将在体外测定。

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.