Transcription is the major control point of gene expression and RNA polymerase (RNAP) is the central enzyme of transcription. Our long term goal is to understand the mechanism of transcription and its regulation. This is best accomplished with prokaryotic RNAPs, mainly because of the high degree of conservation of structure and function from bacteria to man. To this end, we determined the 3.3 Angstrom-resolution X-ray crystal structure of core RNAP from Thermus aquaticus (Taq). Despite this breakthrough, the structure/function relationship of the key transcriptional regulatory molecule in bacteria, the promoter-specificity sigma subunit, remains a major unsolved problem. In the previous funding period, we solved the 2.6 Angstrom crystal structure of a protease-resistant domain of E. coli sigma70 containing almost all of region 2, which is responsible for recognition of the conserved -10 hexamer of the promoter (the Pribnow box) and is involved in melting the double-stranded DNA to form the open promoter complex. This competing continuation focuses on the structural and functional characterization of additional bacterial a factor domains. More specifically, we propose to: 1. Solve crystal structures of sigma factor domains. We have obtained promising crystals of domains of Taq sigma- A that include regions 1.2-3.1 and region 4.1-4.2 (responsible for recognition of the conserved -35 hexamer of the promoter, and also the target for a number of transcription activators; 2. Structurally characterize the sigma/anti-sigma/anti-anti-sigma (sigmaF/SpoIIAB/SpoIIAA) regulatory system from Bacillus stearothermophilus. We have cloned, expressed, purified, and characterized these regulatory factors, and have obtained preliminary crystals of the sigmaF:SpoIIAB complex; 3. Functionally characterize sigma factor domains using a combination of segmental labeling and NMR techniques. We will study intermolecular interactions between the domains of Thermotoga maritima sigmaA (proposed to be involved in the autoinhibitory mechanism preventing sigma binding to promoter DNA in the absence of RNAP), as well as intramolecular interactions with promoter DNA, transcription factors, and RNAP subunits.
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