This application proposes to extend detailed studies of the E. coli RNA polymerase major sigma subunit (sigma-70), the interaction of sigma-70 with the core polymerase subunit beta prime, and the competition of various members of the sigma family for binding to core. Specifically, the aims are: To study the interaction of core polymerase subunit beta prime with sigma-70. We have recently developed a powerful new method (ordered fragment ladder far-Western analysis) for mapping protein-protein interaction domains and have used it to map a major binding site for sigma-70 to the amino acid 260-309 region of the beta prime subunit. We will continue these studies to determine the precise nature of this interaction of beta prime 260-309 region of the beta prime subunit. We will continue mutagenesis studies and determine the structure of beta prime 260-309 by NMR in the presence and absence of sigma-70. We will determine where on sigma-70 this interaction is occurring and test the hypothesis that homologous regions of other sigma family members interact with this same site on beta prime. To study competition of sigma-family members for core. We have overproduced and purified all seven E. coli sigmas and have developed immunological reagents and methods to accurately quantitate them. We will measure the binding parameters of each sigma for core under several ionic conditions. We will study the competition of these various sigmas for core polymerase in purified in vitro systems. We will use our binding results to model the multiple interactions among sigmas and core. We will perturb in vivo conditions by inducing moderate overproduction of a sigma and then determine the effect of this perturbation on the expression of genes under the control of each of the seven sigmas using DNA microarrays. In this way we will test the hypothesis that global regulation of transcription is determined by sigma competition for core. To explore the medical implications of our results. Detailed knowledge of the interactions of sigma-70 and other sigmas with core has potential medical applications. A long-term goal of our studies is to learn enough to devise specific small molecules that will interfere with these key interactions. We will identify peptides and/or small molecules that bind to and interfere with sigma-core binding. This might lead to the development of a new class of antibiotics.
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