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 Daltons, and addition of the auxiliary factors doubles both the number of polypeptides and the protein mass.
Specific aims for the coming project period are as follows: 1. X-ray structure determination of RNA polymerase II at atomic resolution. Two new crystal forms of the enzyme, diffracting to 3.1 and 2.9 Augstrom units resolution, as well as a phase set at better than 4 Augstrom units resolution currently in hand, promise the early accomplishment of this aim. Several approaches will be taken, including the collection of additional derivative data, crystal averaging, and improved processing of existing data. 2. X-ray structure determination of RNA polymerase II complexed with DNA, RNA, DNA and RNA (elongation complex), TFIIB, TFIIE, TFIIS, Rpb4, Rpb7, and -amanitin at atomic-resolution. Datasets have been obtained from five of the nine crystal types with acceptable processing statistics to 3.5 Augstrom units resolution or better. Molecular replacement solutions will be sought, or derivatives will be prepared and X-ray data collected with the use of the derivatives previously employed. 3. Crystallization and structure determination of an arrested RNA polymerase II transcription complex. Recent studies of E. coli RNA polymerase elongation complexes have revealed an oscillation of the enzyme between transcriptionally active and inactive states, conductive to RNA chain elongation and RNA cleavage/proofreading respectively. Our current crystallographic studies of a paused (active) RNA polymerase II elongation complex will be extended to an arrested (inactive) complex. 4. Crystallization of Scizosaccharomyces pombe and human RNA polymerases. Preliminary results suggest the feasibility of extending our studies to these important enzymes. They are likely to be more closely related to one another than to the enzyme from Saccharomyces cerevisiae, the object of our current investigations.
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| Guan, Shenheng; Trnka, Michael J; Bushnell, David A et al. (2015) Deconvolution method for specific and nonspecific binding of ligand to multiprotein complex by native mass spectrometry. Anal Chem 87:8541-6 |
| Fazal, Furqan M; Meng, Cong A; Murakami, Kenji et al. (2015) Real-time observation of the initiation of RNA polymerase II transcription. Nature 525:274-7 |
| Azubel, Maia; Koivisto, Jaakko; Malola, Sami et al. (2014) Nanoparticle imaging. Electron microscopy of gold nanoparticles at atomic resolution. Science 345:909-12 |
| Liu, Xin; Bushnell, David A; Kornberg, Roger D (2013) RNA polymerase II transcription: structure and mechanism. Biochim Biophys Acta 1829:2-8 |
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