Proteomics of RNA polymerase interactomes in pathogenic bacteria

描述(由申请人提供)：本提案结合质谱学、遗传学和计算方法来阐明致病细菌金黄色葡萄球菌和炭疽杆菌中基因表达的中心酶-RNA聚合酶的瞬时相互作用，以及由该酶在体内形成的更稳定的大分子复合体的拓扑和组成。在这些生物体中已经发现了几种毒力调节因子，但它们的机制、辅助因子和修饰酶(如激酶或磷酸酶)仍然很不清楚。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想的转录因子，以及一些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个主要的炭疽毒力因子Atxa，一个金黄色葡萄球菌Tex(用于毒素表达)样因子YhgF，以及几个功能未知的蛋白质。我们建议将这项工作扩展到对RNA聚合酶相互作用因子(相互作用组)进行全面的表征，以确定潜在的毒力调节因子及其辅助因子和修饰酶，并阐明它们在体内的复合体的组成和拓扑结构。我们将使用炭疽杆菌和金黄色葡萄球菌菌株，它们被设计成表达基因的基因组副本，编码亲和力标记的RNA聚合酶亚单位和感兴趣的转录因子，以分离它们的天然复合体，并通过质谱仪表征它们的组成。我们将探索各种生长条件，包括那些诱导毒力因子表达的条件，并使用各种技术来捕获和丰富瞬时相互作用。因此，我们将对RNA聚合酶相互作用组和关键转录调控因子的相互作用进行全面的调查，发现新的转录因子，并对已知转录因子的机制提供深入的了解。通过如上所述在体内进行交联和分离亲和标记的复合体，我们将获得具有辅助因子的共价稳定的RNA聚合酶复合体的“快照”。使用以前列举的相互作用作为搜索空间，分子间的交联链将被“映射”(交联链的位置和交联肽的身份将被确定)(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建立因子的同源模型，通过应用从“映射”数据获得的空间约束来生成络合物的结构模型(通过分子对接方法，如黑线鳕)。否则，我们将对这些数据进行处理，以阐明结构不可用且不能高度可信地模拟的络合物的组成和拓扑。综上所述，这项研究将促进我们对炭疽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分析的建立，有助于发现新的转录因子及其辅助因子和活性调节因子，为已知毒力调节因子的作用机制和结构提供洞察，并鉴定新的毒力调节因子。

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.