Our long term goal is to understand the structural and functional organization of E. coli RNA polymerase. Such knowledge is crucial to understanding how RNA polymerase performs elementary steps in transcription and prerequisite for a molecular understanding of how small molecule and protein factors regulate RNA polymerase activity. The selection and analysis of mutations is an excellent point of entry to this problem. Intensive analysis of particular mutant classes defines limited areas of the protein, and characterization of the in vivo phenotypes suggests the likely functional alterations. These results can then be used to design experiments to determine if the mutational defect affects transcription in vitro in the way suggested by in vivo analysis and to demonstrate directly that a particular region of protein carriers out the proposed function. Thus our specific aims are: 1. The role of the sigma subunit in determining promoter specificity. We will analyze the effects of sigma 70 mutants on kinetics of promoter recognition select mutations in sigma 32 with altered promoter recognition, and make sigma 70-sigma 32 hybrids to localize the region of sigma interacting with the -10 region of the promoter. We will determine if intact sigma 70 and sigma 32 and portions of each of these sigma's bind to promoter DNA. If successful, we will design a partial sigma that acts as DNA binding molecule in vivo, for structural and mutational analysis. 2. To analyze the molecular basis for the pleiotropic effects of Rifr mutations. We will determine whether selected Rifr mutations affect various steps in RNA synthesis which can be monitored in vitro, including: formation of the DNA/RNA hybrid region in the transcription bubble, RNA affinity to the beta subunit, K/M's for nucleotide binding, abortive initiation and pausing; create an antibody population which is specific for the major Rif domain to enable us to monitor whether this region is involved in the binding of rifampicin, substrate or nascent RNA and map and characterize allele specific second site revertants of Rifr mutations to probe for regions of beta and beta' which lie in close proximity to the Rif domain. 3. To obtain mutations that affect specific functions of RNA We will improve the genetic technology for selecting RNA polymerase mutations, and, by selecting, mapping and analyzing mutations, locate regions of RNA polymerase involved some of the following functions: nucleotide binding, translocation, NusA binding, and termination.
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