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 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 DNA RNAProtein

Prokaryotic transcription RNA polymerase containing 5 subunits One polymerase for all transcription  -factor recognize promoter region on the DNA

Eukaryotic 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 transkription

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 how the pol II enzyme of yeast and, by implication, of other higher organisms performs its critical role." Jean Marx Science (2001), Apr20,

Xray structure determination Protein crystals Collecting data Determination of the structure

Xray structure determination Collecting data

Xray structure determination Result Electron density map of the structure Determination 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,

RNA Polymerase II (yeast)

RNA polymerase II

RNA polymerase II in action

Inhibitor of RNA polymerase II

Binding of  -amanitin to the active site of RNA polymerase II

”Proofreading” of an incoming nucleotide

Close-up view of ”proofreading” (matched nucleotide, UTP)

Close-up view of ”proofreading” (mismatched nucleotide, ATP)

Overlay matched/mismatched nucleotide

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

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