This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of the proposed research is to determine the X-ray structures 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 Dalton, and addition of the auxiliary factors doubles both the number of polypeptides and the protein mass. Solution of the problem requires a combination of biochemical studies, electron and X-ray crystallography. Eight types of RNA polymerase II crystal are now in hand, crystals of the native enzyme, and cocrystals with DNA, with RNA, with both DNA and RNA in the transcriptionally active state, with TFIIB, with TFIIE, with the additional polymerase subunits RPB4 and RPB7, and with the tight-binding inhibitor a-amanitin. Recent progress includes a low resolution envelope and phases to 6.5 and the recent collection of several novel heavy atom cluster complexed with RNA Pol II. The prognosis is now very good for the rapid achievement of a high resolution structure.
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