Our fundamental interest is the central biological problem of how the cell regulates transfer of information from gene to protein. RNA polymerase not only carries out transcription, but also integrates intracellular and extracellular signals by adjusting its transcriptional potential to the sum of the regulatory inputs. To understand how various cellular regulatory systems control RNA polymerase, our approach has been to first delineate the functional anatomy of the enzyme and then investigate how altering its individual functions affects gene regulation. We continue this approach with a focus on how the interaction between RNA polymerase and its initiation factors conveys information to each partner. In addition, we initiate a new approach: genome-wide analysis. Following perturbations that alter the interaction of initiation factors and regulators with RNA polymerase, we will be able, for the first time, to correlate functional changes in RNA polymerase with global changes in gene expression. During the current granting period we will: 1. Identify the roles of sigma 70-core contact sites in RNA polymerase function 2. Use minimal assemblies to identify the functions of contacts between sigma 70 and core. 3. Test the sequential interaction model for the interface between sigma and core. 4. Further define the interface between sigma 70 and core. 5. Determine global effects of transcriptional apparatus alterations using microarray analysis. These studies provide a paradigm for how initiation factors and RNA polymerase prepare each other for initiation in all prokaryotes, including pathogens. Moreover, given the extensive structural conservation between prokaryotic and eukaryotic RNA polymerase, especially near the active site, our findings are likely to have general applicability to all organisms. Finally, our studies on the circuitry governing expression in Escherichia coli will be a useful blueprint as attention switches to newly determined genomic sequences of pathogens lacking the rich history of genetic and biochemical investigation that is enjoyed by E. coli.
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