We propose to study in detail the structure of the Escherichia coli RNA polymerase major sigma subunit(sigma70),the interaction of sigma70 with core polymerase subunits, and the additional members of the sigma family and how they compete for binding to core. 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. Specifically, our aims are: A. To Study the Structure and Function of Sigma-70. We will test our hypothesis that sigma70 forms a """"""""hairpin"""""""" structure and must open or partially unfold to function. We will crystallize sigma70, determine its 3-D crystal structure, and compare its structure to other sigmas. We will use mutations and protein fragments to probe functional domains utilizing a rapid method for producing proteins and protein fragments in Escherichia coli S-30 extract systems. We will determine what step in the transcription process each MAb to sigma70 is inhibiting. We will more precisely epitope map and study several particularly interesting MAbs. B. To Identify Regions of Core Polymerase Subunits Involved in Sigma Interaction. We will use chemical crosslinking and specific MAbs to determine the site of binding of sigma70 and a short 25 amino acid region of sigma70 to core. We will also determine the regions on core involved in interactions with other sigmas. C. To Study Competition of Sigma-Family Members for Core. We will measure the in vivo levels of known sigmas as a function of growth conditions. We will study the competition of these various sigmas for core polymerase both in vivo and in purified in vitro systems. We will examine the role of anti-sigmas in affecting sigma competition. D. To Study Sigma-S and Sigma-70 Holoenzyme Interaction with the bolAp1 Promoter. We will determine the interactions of holoenzyme containing sigma70 or sigmaS with the bolAp1 promoter and determine why both holoenzymes function in vitro but only E-sigma-S functions in vivo at this promoter. We propose to continue to study sigma structure and function by a concerted use of biochemistry, protein and physical chemistry, MAb. technology, and molecular genetics. Our long experience studying sigma and our recent progress place us in a unique position to carry out the proposed experiments and to answer many important remaining questions about the structure and function of Escherichia coli sigmas.
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