Structure of RNA Polymerase II

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DESCRIPTION (provided by applicant): The goal of the proposed research is to determine the X-ray structure of RNA polymerase II and of its complexes with nucleic acids and auxiliary protein factors at atomic resolution. The problem is challenging, since the polymerase alone comprises 15 polypeptides with a total mass of nearly 600,000 Daltons, and addition of the auxiliary factors more than doubles both the number of polypeptides and the protein mass. The proposed research represents the culmination of our efforts, and will provide a basis for understanding a vast body of biochemical and genetic data on transcription accumulated in diverse systems over the past 30 years. Specific aims for the next project period are as follows: 1. Reveal the exit path of RNA from an RNA polymerase II transcribing complex. We will determine the X-ray structure of a new crystal containing a 34-residue transcript. 2. Undertake the structural analysis of large, multicomponent transcription elongation complexes. We will attempt to crystallize and solve the structure of RNA polymerase II - Spt4/Spt5 complexes, as a step towards the structure determination of the entire P-TEFb/NELF/DSIF system, which has particular clinical, as well as fundamental significance. 3. Elucidate the mechanism of RNA polymerase II start site selection. We will pursue recent findings concerning the structure of the "B finger" domain of general transcription factor TFIIB and its role in start site selection. We propose to determine the X-ray structures of RNA polymerase II complexes with the B finger alone, with the B finger and a strand of template DNA, and with the B finger, a strand of template DNA, and a 5-residue RNA "transcript." 4. Determine the structures of RNA polymerase II - general transcription factor complexes, with the ultimate goal of solving the entire RNA polymerase II transcription initiation complex. We will attempt to crystallize and solve the structures of RNA polymerase II complexes with TFIIE, with TFIIF, with TFIIE and TFIIH, with a TBP - TFIIB - promoter DNA complex, with TBP - TFIIB - Tfg2ffFIIF - promoter DNA complexes, and with all components, including promoter DNA in both "closed" and "open" states.