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 determining 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 (sigma70), the interactions of sigma70 with the core polymerase and specific promoter DNA sequences, and the additional members of the sigma family. In this way we hope to understand how sigma70 functions to determine the selectivity of RNA polymerase binding and RNA chain initiation, and how the cell can alter its pattern of transcription by using other sigma-like factors. 1) We will prepare and characterize monoclonal antibody (MAb) reagents needed to study E. coli transcription machinery. We will prepare MAbs to several recently discovered sigmas (sigmaE, sigmaF, sigmaK). We have developed a more effective method for rapid epitope mapping and will use it to fully characterize our present and future MAbs. We will continue to improve our immunoaffinity purification procedures. These MAbs are essential for us to carry out the quantitation of subunits in vivo, localization of subunit-subunit crosslink sites, probing of sigma70 structure and function, and rapid purification proposed in this grant. 2) We will continue our study of the structure and function of sigma70. We will study the site of binding of sigma70 to core polymerase. We will determine what step in the transcription process each MAb to sigma70 is inhibiting. We have developed a rapid method for producing protein fragments and will use it to characterize functional domains of sigma70. We will collaborate on 2-D and 3-D crystal structure determination. 3) We will study holoenzymes, particularly the factors affecting competition of sigma-family members with core. We will purify milligram amounts of the known sigmas for core polymerase both in purified systems in vitro and in vivo. We will determine the regions on core involved in interactions with various sigmas.
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