Regulation of the initiation of RNA synthesis is now known to be a major mechanism for regulating gene expression and thus the enzymatic capabilities of a cell or organism. Many disorders are the result of faulty regulation of gene expression. It is important therefore to understand the basic mechanism of transcription and its control. Considerable progress has been made in defining the transcriptional machinery in bacteria, in particular the subunits of RNA polymerase. The sigma subunit is known to be important in determing the selectivity of RNA chain initiation in vitro and in vivo. We propose to study in detail the structure of the E. coli RNA polymerase major sigma (sigma-70) and the interactions of sigma with the core polymerase and specific promoter DNA sequences. In this way we hope to understand how sigma functions to determine the selectivity of RNA polymerase binding and RNA chain initiation, and how the cell can alter its trascription pattern by using other sigma-like factors. 1) We will use and improve a number of powerful research tools in our studies, including: our ability to prepare large amounts of pure active sigma, monoclonal antibodies to sigma, sophisticated computer analysis, a peptide map for use in locating sites of modification and crosslinks along the sigma polypeptide, and our ability to synthesize oligonucleotides to construct mutant promoters and carry out site-directed mutagenesis of sigma. 2) We will use the best computer methods available to make secondary structutr predictions from the sigma sequence and to compare sequences of homologous sigma-like factors to determine conserve regions of the molecule. 3) We will use chemical modification, protein-protein and protein-DNA cross-linking, protease susceptibility, and immunological reagents to probe the structure of free sigma and its interactions with core polymerase and promoter DNA. 4) We will use monoclonal antibodies we have already prepared to determine cross-reactivity of sigmas in other prokaryoted and of other E. coli sigma-like proteins and to purify them by immunoaffinity chromatography. 5) We will continue our studies of the regulation of the rpsU-dnaG-rpoD operon.
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