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、遗传学和计算方法，以阐明致病性细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确定了少数毒力调节因子，但它们的机制，辅因子，修饰酶（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种“孤儿”调节因子，基于与来自其他生物的已知因子的同源性，这些调节因子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。在一系列的初步实验中，我们已经在RNA聚合酶结合蛋白中鉴定出一种主要的炭疽毒力因子AtxA。aureus 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进行全面的表征RNA聚合酶相互作用因子（相互作用组），以确定潜在的毒力调节剂和他们的辅助因子和修饰酶，并阐明其复合物的组成和拓扑结构在体内。 我们将使用B菌株。anthracis和S.金黄色葡萄球菌，工程化以表达基因的基因组拷贝，编码感兴趣的RNA聚合酶和转录因子的亲和标记的亚基，以分离它们的天然复合物并通过质谱法表征它们的组成。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，我们将获得一个全面的调查RNA聚合酶的相互作用，和相互作用的关键转录调节因子，确定新的转录因子，并提供深入了解已知的机制。 通过进行如上所述的体内交联和分离亲和标记的复合物，我们将获得具有辅助因子的RNA聚合酶复合物的共价稳定的“快照”。将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）来“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和建立因子的同源性模型，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，如HADDOCK）。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，并且不能以高置信度建模。 总之，这项研究将促进我们对B基因表达的理解。anthracis和S.包括毒力因子在内的金黄色葡萄球菌的基因组学研究，有助于建立这些病原体的体外转录测定，有助于发现新的转录因子、它们的辅因子和活性调节因子，为已知毒力调节因子的作用提供了机制和结构上的见解，并鉴定了新的毒力调节因子。 公共卫生关系：该提案旨在对致病菌金黄色葡萄球菌和炭疽杆菌中参与基因表达及其调控的蛋白质因子进行全面的质谱询问，以提供已知毒力调节剂的结构和机制见解，并鉴定新的毒力调节剂。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点，包括毒力调节因子，其辅助因子和修饰酶。