Considerable progress has been made in the last decade in defining the transcription machinery in bacteria, in particular the subunits of RNA polymerase. However, the precise function and properties of the sigma subunit, known to be important in determining the selectivity of RNA chain initiation in vitro, are far from understood. We propose a detailed study of the structure and function of sigma: 1) We will construct a map of sigma by sequencing the cloned gene and correlating it with peptides obtained by selectively cleaving the polypeptide chain with a variety of cleaving reagents and enzymes. Comparison of sigmas from various bacterial strains should indicate whether common features exist. We will locate on this map, by crosslinking, chemical modification, affinity chromatography, and fine structure genetic mapping of mutants, the structural and functional domains of sigma essential for blinding to core polymerase, for selectivity of promoter binding and initiation, and for possible interactions with other regulatory molecules. 3) We have recently characterized and mapped two mutations of E. coli K12 with thermosensitive sigma activity and will use them and other sigma mutations to identify the in vivo consequences of the mutations. We will use second site revertants of sigma mutations to probe interaction of sigma with other regulatory componenets of the cell. We will use a variety of assays to determine whether the pattern of selectivity of promoter binding and initiation of transcription in vitro is altered in any of these mutants. 3) We will study the regulation of sigma synthesis and degradation in vivo and in vitro. The structure of the sigma operon including regulatory regions such as promoters, attenuators, and terminators will be determined.
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