250 likes | 349 Views
Nobel Prize in Chemistry 2006 Roger D Kornberg Molecular basis of eukaryotic transcription. Summary of his contributions. ~75 published articles last 10 years Developed a yeast system for production and purification of RNA polymerase II
E N D
Nobel Prize in Chemistry 2006Roger D KornbergMolecular basis of eukaryotic transcription
Summary of his contributions • ~75 published articles last 10 years • Developed a yeast system for production and purification of RNA polymerase II • Discovery of the Mediator complex in eukaryotic transcription • Structure determination of more than a dozen new crystal structures of RNA polymerases with DNA, RNA inhibitors and protein complexes
Central Dogma RNA Protein DNA
RNA polymerase containing 5 subunits One polymerase for all transcription s-factor recognize promoter region on the DNA Prokaryotic transcription
RNA polymerases I,II and III RNA polymerase II, 12 subunits, MW~0.5MDa 5 transcription factors, TFIIB,E,F,H and TBP Mediator complex Eukaryotic transcription
Structure of RNA polymerase II "If any enzyme does the cell's heavy lifting, it's RNA polymerase II. Its job: getting the synthesis of all the proteins in higher cells under way by copying their genes into RNAs, and doing it at just the right time and in just the right amounts. As such, pol II, as the enzyme is called, is the heart of the machinery that controls everything that cells do--from differentiating into all the tissues of a developing embryo to responding to everyday stresses. Now, cell biologists can get their best look yet at just howthe pol II enzyme of yeast and, by implication, of other higher organisms performs its critical role." Jean Marx Science (2001), Apr20, 411-414
Xray structure determination • Protein crystals • Collecting data • Determination of the structure
Xray structure determination • Collecting data
Xray structure determination • Determination the structure Result Electron density map of the structure
Structure determination of RNA polymerase II Resolution down to 2.8 Å 10 subunits ~3500 amino acid residues Collected data on synchrotron Large unit cell (a=122.7 Å,b=223.0 and c=376.1) The ”core” consist of RpbI (green and RpbII (cyan)
Illustrations from: Aaron Klug, Science 2001, 292, 1844-1846
Binding of a-amanitin to the active site of RNA polymerase II
Close-up view of ”proofreading” (mismatched nucleotide, ATP)
Concluding remarks • Molecular understanding of promoter recognition • Mechanism for initiation, translocation • Mechanism for selection of an incoming ribonucleotide complementary to template DNA • Future: Role of transcriptionfactors and Mediators in regulation