Our fundamental interest is a central biological problem -- how does the cell regulate transfer of information from gene to protein? We focus on RNA polymerase, the enzyme responsible for transcription. We view this enzyme as the ultimate target of a regulatory system that adjusts transcription in response to intracellular and extracellular signals by modulating the enzymatic functions of RNA polymerase. To understand this regulatory system, we first identify regions of RNA polymerase involved in a particular function by selecting, mapping and sequencing particular classes of mutants, or where appropriate by crosslinking. We then investigate how altering individual functions affects gene regulation. Because the subunit structure of RNA polymerase is highly conserved, our structure-function analysis, which is most feasible in E. coli, should have broad applicability. In the current granting period we will: 1. Further define the role of sigma in transcription initiation We will select mutations in sigma32 that block initiation in vivo in the presence of the wild type sigma32 and determine whether the mutants are defective in strand opening, phosphodiester bond formation or sigma release. In addition, we will further delineate the promoter recognition properties of sigma by determining which mutational changes in promoter DNA affect recognition by fragments of sigma70 and assessing the effects of alanine substitution mutations in sigma32 on promoter recognition. 2. Define the interactions between sigma and core RNA polymerase We will select point mutants in sigma32 and sigma70 that are defective in binding to core RNA polymerase and use them to obtain compensatory mutations in core RNA polymerase. Mutants will be characterized for their functional and regulatory effects. 3. Define functional domains in core RNA polymerase a) interaction with NusA. To define the site(s) on polymerase that bind NusA and allow us to further determine the role(s) of NusA in vivo, we will screen a group of RNA polymerase mutants defective in NusA dependent termination for those defective in binding or responding to NusA. b) substrate binding and translocation. To further characterize the regions that comprise the active site of RNA polymerase, we will identify and characterize in vitro mutants that are resistant to streptolydigin and mutants that elongate more slowly than wild-type. c) binding to DNA. We will use chemical crosslinking to identify regions of core RNA polymerase close to DNA during initiation, transition to elongation and elongation.
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