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: 8339433
• 负责人: EVGENY A NUDLER
• 金额: $ 21.13万
• 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 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8339433
• 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.

描述（由申请人提供）：该提案结合了质谱、遗传和计算方法，以阐明致病菌金黄色葡萄球菌和炭疽芽孢杆菌中基因表达的中心酶RNA聚合酶的瞬时相互作用，以及该酶在体内形成的更稳定的大分子复合物的拓扑结构和组成。在这些生物体中已经鉴定出少数毒力调节因子，但它们的机制、辅助因子、修饰酶（例如激酶或磷酸酶）仍然很大程度上不清楚。此外，这些病原体的基因组编码数百个“孤儿”调节因子，根据与其他生物体已知因子的同源性，被注释为假设的转录因子，以及许多没有预测功能的蛋白质。在一系列初步实验中，我们在 RNA 聚合酶结合蛋白中鉴定出了主要的炭疽毒力因子 AtxA、金黄色葡萄球菌 Tex（用于毒素表达）样因子 YhgF 和几种功能未知的蛋白质。我们建议扩大这项工作，对RNA聚合酶相互作用因子（相互作用组）进行全面表征，识别潜在的毒力调节因子及其辅助因子和修饰酶，并阐明其体内复合物的组成和拓扑结构。 我们将使用炭疽芽孢杆菌和金黄色葡萄球菌菌株，这些菌株被设计为表达基因的基因组拷贝，编码RNA聚合酶和感兴趣的转录因子的亲和标记亚基，分离它们的天然复合物并通过质谱分析表征它们的组成。我们将探索各种生长条件，包括诱导毒力因子表达的条件，并采用各种技术来捕获和富集瞬时相互作用。因此，我们将获得对 RNA 聚合酶相互作用组和关键转录调节因子相互作用组的全面调查，识别新的转录因子并提供对已知转录因子机制的见解。 通过如上所述进行体内交联和分离亲和标记复合物，我们将获得具有辅助因子的RNA聚合酶复合物的共价稳定“快照”。使用先前列举的相互作用组作为搜索空间（减少计算成本和时间），将“映射”分子间交联（将确定交联的位置和交联肽的身份）。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，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（通过分子对接方法，例如 HADDOCK）。否则，我们将处理这些数据以阐明复合物的组成和拓扑结构，这些结构不可用且无法高置信度地建模。 总而言之，这项研究将增进我们对炭疽芽孢杆菌和金黄色葡萄球菌基因表达的理解，包括毒力因子的基因表达，促进这些病原体的体外转录测定的创建，帮助发现新的转录因子、它们的辅助因子和活性调节因子，为已知毒力调节因子的运作提供机制和结构见解，并鉴定新的毒力调节因子。