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

DNARNA Protein

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, 411-414

Xray structure determination

•Protein crystals

•Collecting data

•Determination of the structure

Xray structure determination

•Collecting data

Xray structure determination

ResultElectron 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, 1844-1846

RNA Polymerase II (yeast)

RNA polymerase II

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

Thanks for listening.....