Proteomics of RNA polymerase interactomes in pathogenic bacteria

病原菌 RNA 聚合酶相互作用组的蛋白质组学

• 批准号: 8173472
• 负责人: EVGENY A NUDLER
• 金额: $ 25.35万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-28 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8173472
• 关键词: Affinity; Affinity Chromatography; Anthrax disease; Architecture; Bacillus anthracis; Binding Proteins; Biochemical; Biological Assay; Cells; Chemicals; Classification; Code; Complex; DNA-Directed RNA Polymerase; Data; Docking; Drug Delivery Systems; Drug Design; Elastin; Engineering; Enzyme Gene; Enzymes; Formaldehyde; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genome; Genomics; Growth; Homology Modeling; In Vitro; Macromolecular Complexes; Maps; Mass Spectrum Analysis; Measures; Methods; Modeling; Molecular; Organism; Orphan; Pathway interactions; Peptides; Phosphoric Monoester Hydrolases; Phosphotransferases; Positioning Attribute; Precipitation; Protein Kinase; Proteins; Proteomics; Regulation; Research; Series; Set protein; Staphylococcus aureus; Structural Models; Structure; Surveys; Techniques; Time; Toxin; Virulence; Virulence Factors; Work; base; computerized data processing; computerized tools; cost; crosslink; his6 tag; in vivo; insight; interest; leucylmethionine; macromolecular assembly; novel; operation; pathogen; pathogenic bacteria; research study; transcription factor

DESCRIPTION (provided by applicant): This proposal combines mass-spectrometric, genetic, and computational approaches to illuminate the transient interactions of the central enzyme of gene expression, RNA polymerase, in pathogenic bacteria Staphylococcus aureus and Bacillus anthracis, as well as the topology and composition of the more stable macromolecular complexes formed by this enzyme in vivo. A handful of virulence regulators have been identified in these organisms, but their mechanisms, co-factors, modifying enzymes (such as kinases or phosphatases) remain largely obscure. In addition, the genomes of these pathogens encode hundreds of "orphan" regulators, annotated as hypothetical transcription factors based on homology to known factors from other organisms, and a number of proteins with no predicted function. In a series of pilot experiments we have identified among RNA polymerase-binding proteins a major anthrax virulence factor, AtxA, a S. aureus Tex(for toxin expression)-like factor YhgF, and several proteins of unknown function. We propose to expand this work to carry out a comprehensive characterization of RNA polymerase-interacting factors (interactome), to identify potential virulence regulators and their co-factors and modifying enzymes, and to elucidate the composition and topology of their complexes in vivo. We will use strains of B. anthracis and S. aureus, engineered to express genomic copies of the genes, coding for affinity-tagged subunits of RNA polymerase and transcription factors of interest, to isolate their native complexes and characterize their composition by mass-spectrometry. We will explore a variety of growth conditions, including those where virulence factors expression is induced, and employ various techniques to trap and enrich for transient interactions. As a result we will have obtained a comprehensive survey of RNA polymerase interactome, and interactomes of the key transcription regulators, identifying new transcription factors and providing insights into the mechanisms of the known ones. By performing in vivo cross-linking and isolating affinity tagged complexes as described above, we will obtain covalently stabilized "snap-shots" of RNA polymerase complexes with accessory factors. Intermolecular cross-links will be "mapped" (position of the cross-link and identity of cross-linked peptides will be determined) using previously enumerated interactomes as the search space (reducing the computational cost and time). Whenever possible we will use available structural information and build homology models of the factors to generate structural models (via molecular docking approaches such as HADDOCK) of the complexes by applying spacial constrains obtained from the "mapping" data. Otherwise we will process these data to elucidate composition and topology of the complexes which structures are not available and can not be modeled with high confidence. Taken together this research will advance our understanding of gene expression in B. anthracis and S. aureus, including that of virulence factors, facilitate creation of the in vitro transcription assays for these pathogens, aid the discovery of new transcription factors, their co-factors and regulators of activity, provide mechanistic and structural insights into the operation of known virulence regulators, and identification of novel ones. PUBLIC HEALTH RELEVANCE: This proposal aims at comprehensive mass-spectrometric interrogation of protein factors involved in gene expression and its regulation in pathogenic bacteria Staphylococcus aureus and Bacillus anthracis, to provide structural and mechanistic insights into operation of known virulence regulators, and identification of the new ones. This research will advance our understanding of gene expression mechanisms in these pathogens, aid in genome annotation and strain classification, and generate a new set of potential drug targets, comprising regulators of virulence, their co-factors and modifying enzymes.

描述（由申请人提供）：该提案结合了质谱，遗传和计算方法，以阐明基因表达的中心酶RNA聚合酶在致病细菌金黄色葡萄球菌和炭疽菌的葡萄球菌中的瞬时相互作用，以及通过拓扑结构和拓扑结构的质量和组成，以及这种相互稳定的组合。在这些生物体中已经鉴定出了少数毒力调节剂，但是它们的机制，辅助因子，修饰酶（例如激酶或磷酸酶）仍然很大程度上晦涩难懂。此外，这些病原体的基因组编码了数百个“孤儿”调节剂，这是基于与其他生物体的已知因素的同源性的假设转录因子，以及许多没有预测功能的蛋白质。在一系列的试验实验中，我们已经在RNA聚合酶结合蛋白中鉴定出主要的炭疽毒力因子，ATXA，Aureuss。tex（用于毒素表达） - 样因子YHGF和几种功能不明功能的蛋白质。我们建议扩大这项工作，以对RNA聚合酶相互作用因子（Interactome）进行全面表征，以鉴定潜在的毒力调节剂及其共同因素并修饰酶，并阐明其体内复合物的组成和拓扑。 我们将使用炭疽芽孢杆菌和金黄色葡萄球菌的菌株，设计为表达基因的基因组拷贝，编码RNA聚合酶的亲和力标记的亚基和感兴趣的转录因子，以分离其天然复合物并通过质谱法表征其组成。我们将探索各种生长条件，包括诱导毒力因子表达的那些生长条件，并采用各种技术来捕获和丰富瞬态相互作用。结果，我们将对RNA聚合酶相互作用组和关键转录调节剂的相互作用体进行全面调查，识别新的转录因子并提供有关已知机制的见解。 通过如上所述，通过在体内交联和分离亲和力标记的复合物中，我们将获得具有辅助因子的RNA聚合酶复合物的共价“快照”。分子间的交联将“映射”（交联的位置和交联肽的身份）使用先前列举的相互作用作为搜索空间（减少计算成本和时间）。只要有可能，我们将使用可用的结构信息并构建因子的同源模型，以生成复合物的结构模型（通过分子对接方法，例如黑线径），通过应用从“映射”数据获得的空间约束来生成复合物的结构模型。否则，我们将处理这些数据，以阐明结构不可用的复合物的组成和拓扑结构，并且无法以高信心进行建模。 总结这项研究将提高我们对炭疽芽孢杆菌和金黄色葡萄球菌中基因表达的理解，包括毒力因素的表达，促进为这些病原体的体外转录测定法创建，有助于发现新的转录因子，其共同因素和活动调节剂，为已知的毒品调节剂的机械和结构洞察力提供了机械和结构性洞察力，并识别了新小说和识别的新颖性。 公共卫生相关性：该提案的目的是对涉及基因表达中涉及的蛋白质因子及其在致病细菌中的调节金黄色葡萄球菌和炭疽杆菌的调节，以提供有关已知毒力调节剂的运作以及新的鉴定的结构和机械洞察力。这项研究将促进我们对这些病原体中基因表达机制的理解，有助于基因组注释和菌株分类，并产生一组新的潜在药物靶标，包括毒力的调节剂，其共同因素和修饰酶。