STRUCTURE OF RNA POLYMERASE II

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The long term goal of our research is to elucidate the mechanism and regulation of transcription in eukaryotes. We focus on RNA polymerase II, the enzyme responsible for almost all eukaryotic transcription, whose intricate regulation underlies many cellular and developmental processes. Most regulation occurs at the initiation of transcription, which involves the interaction of RNA polymerase II with a set of general transcription factors at a promoter. RNA polymerase II is itself a large assembly: the yeast enzyme is made up of 15 polypeptide chains, with a mass of nearly 600,000 Daltons. The initiation complex comprises more than 25 polypeptides, with a total mass in excess of 1.2 million Daltons. We wish to determine the three-dimensional structure of the entire initiation complex, as an essential step towards understanding the mechanism and regulation of transcription. We will solve the structure piecemeal: the X- ray structures of the polymerase and general transcription factors will be determined individually, and these structures will be combined on the basis of electron crystallography of polymerase-factor complexes. We propose here the X-ray analysis of RNA polymerase II crystals recently obtained, and the formation and analysis of cocrystals of the polymerase with DNA, RNA, and general transcription factors. The results should apply to RNA polymerases and transcription mechanisms in general, since the polymerases, transcription factors, and aspects of the mechanism and regulation of transcription have been conserved through evolution. Aims and experimental approaches of the proposed research include the following: 1. The X-ray structure of yeast RNA polymerase II will be determined at low (6-12 Angstroms) resolution. Information from electron crystallography will be employed in lieu of phases from heavy atom derivatives. The results will be instrumental in achieving all the further objectives of the project. 2. The X-ray structure of yeast RNA polymerase II will be determined at atomic resolution. Heavy atom cluster compounds will be used to prepare derivative crystals, and the locations of the heavy atoms will be found with the aid of the low resolution polymerase structure. 3. Cocrystals of RNA polymerase II with DNA and RNA oligonucleotides will be prepared and analyzed by difference Fourier methods. Analysis initially at low resolution will be sufficient to reveal the template and product binding sites of the enzyme. "Elongation" complexes, containing the enzyme in the act of transcription, will also be crystallized and subjected to structure determination. 4. Cocrystals of RNA polymerase II with a complex of subunits RPB4 and RPB7 and with general transcription factor e (TFIIB) will be prepared. Again initial analysis at low resolution will suffice to reveal sites of interaction with the polymerase.