Proteomics of RNA polymerase interactomes in pathogenic bacteria

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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.