# Presentation: 759724

## Slide 1

## Acknowledgements

## HATs

**HATs**
**HMTs**
**SWI/SNF**
**RSC**

## HATs

**HATs**
**HMTs**
**SWI/SNF**
**RSC**

## Pol II

**the central dogma of molecular biology**

**Pol I**

**Pol II**

**Pol III**

**rRNA**

**tRNA**

**Ribosome**

**DNA**

**mRNA**

**Protein**

**Notes:**
The central dogma of molecular biology reminds us of the central role of RNA polymerase II, or pol II, in the process of gene expression in higher organisms. The first step in gene expression, the transcription of DNA to produce RNA, is carried out by three different RNA polymerases. RNA polymerases I and III transcribe only a few dozen genes to produce mainly ribosomal- and transfer-RNAs, respectively, which are then used to build and fuel the ribosome. In contrast, pol II transcribes the vast majority of genes, all those coding for proteins, to synthesize messenger-RNA which serves as a template for protein synthesis in the ribosome. Pol II is the endpoint of signal transduction pathways. It is the temporal and spatial regulation of pol II that underlies all cell growth and development. 

## RNA polymerase II transcription machinery

**Pol II**

**DNA unwinding**
**RNA polymerization**
**proofreading**

**GTFs**
**(TFIIB, -D, -E, -F, -H)**

**promoter recognition**

## Slide 7

## Slide 8

## Conserved Mediator subunits

| yeast | Drosophila | human |
| --- | --- | --- |
| Med1 | Trap220* | TRAP220 |
| Med4 | Trap36 | TRAP36 |
| Med6 | Med6 | hMed6 |
| Med7 | Med7* | hMed7 |
| Med8 | Arc32* | hNut2 |
| Cse2/Med9 | CG5134* | MED9 |
| Nut2/Med10 | Nut2* | TRAP230 |
| Med11 | Med21 | MED11 |
| Srb8 | Kto* | TRAP240 |
| Ssn2/Srb9 | Skd/Pap/Bli* | TRAP170 |
| Rgr1 | Trap170 | TRAP95 |
| Gal11 | Arc105* | MED15 |
| Sin4 | Trap95* | TRAP80 |
| Srb4 | Trap80 | hTRFP |
| Srb5 | p28/CG14802 | MED18 |
| Rox3 | CG5546* | MED19 |
| Srb2 | Trfp | hSrb7 |
| Srb7 | Trap19 | TRAP150b |
| Srb6 | Med24 | MED22 |
| Med1 | Trap220* | TRAP220 |
| CDK8
cycC | CDK8
cycC | CDK8
cycC |

## Mediator of Transcriptional Regulation

- Required for all transcription of all pol II promoters
- Essential link in the chain of communication: enhancer   activator   Mediator   pol II   promoter
- Co-activator, co-repressor, and general transcription factor
- Processes, transduces transcriptional regulatory information in all eukaryotes

## Slide 11

## 2-D protein crystallization

_**2-D protein crystallization**_

**  ****rapid, small amount of protein (********g)**
**  ****pol II without subunits 4 and 7**
**  ****2-D crystals seed 3-D crystal growth**

## Oxidation

**Crystal polymorphism**
**No standard heavy atom derivatives**

**Obstacles to X-ray structure determination of pol II**

**Notes:**
We have determined the crystal structure of the best studied form of pol II, that from the yeast Saccharomyces cerevisiae. This enzyme comprises 12 polypeptide subunits, and has a total molecular weight of about 1/2 a megadalton. 10 out of the 12 subunits are present in the structure. The structure only lacks 2 small non-conserved subunits.
There is a high degree of sequence conservation throughout the corresponding yeast and human pol II subunits. Moreover, most yeast subunit genes can either fully or partially be replaced by the corresponding human or mouse genes, without loss of viability, showing that they are functionally equivalent. Thus, the structures of yeast and human pol II must be virtually identical.

## Structure determination of RNA polymerase II

- 1971     Lateral diffusion in lipid layers
- 1983     2-D protein crystals on lipid layers
- 1991     2-D crystals seed 3-D crystals
-      Diffraction phased with heavy
-              atom clusters
- 2000     Structure at 2.8 Å resolution

**Structure determination of RNA polymerase II**

## 10 years, 10,000 liters, 1 grad student

**10 years, 10,000 liters, 1 grad student**

## Proteins			10

**Amino acids****	****	****3559****	**
**Atoms****	****	****	****28,378**
**MW****	****	****	****	****0.5 MDa**
**R****free**** (2.8 Å) ****	****	****28.2 %**

## Slide 17

## Template

**Template**

**Hybrid**

**RNA**

**5’**

**Pol II**

**Nontemplate**

## Slide 19

**Notes:**
This is free pol II, viewed from the front into the cleft. A large flexible protein domain that is composed of terminal regions of the two large subunits and stabilized  by 3 zinc ions  is shown in yellow. Here are the structural changes that occur upon formation of the elongation complex. The yellow domain, we call it the clamp, closes upon nucleic acid binding in the cleft. The clamp retains DNA and its closure may account for the observed great stability of the elongation complex.
Downstream DNA, the DNA that is going to be transcribed is pointing towards us. During transcription, pol II moves towards us, the DNA moves inwards. We will look at this again from the top, with pol II rotated by 90 degrees around a vertical axis.

## Slide 20

## Slide 21

## Slide 22

## Slide 23

## post-translocation

**post-translocation**

**NTP enters E site**

**NTP rotates into A site**

**pre-translocation**

## Trigger loop contacts NTP in the A site

**Trigger loop contacts NTP in the A site**

## Multiple conformations of the trigger loop

**Multiple conformations of the trigger loop**

## Trigger Loop Network

**Trigger Loop Network**

## Trigger Loop Network

**Trigger Loop Network**

## Trigger loop couples nucleotide selection to catalysis

**Trigger loop couples nucleotide selection to catalysis**

## Trigger Loop Network

**Trigger Loop Network**

## Slide 31

## Slide 32

## -7

**-7**

**-8**

**-9**

**-10**

- 3.4

- 4.4

- 5.2

- 12 Angstroms

- Template DNA
- RNA

**Notes:**
3.4,4.4,5.2,12

## Fork loop

- Rudder

- Lid

- Template
- RNA

## Structure of pol II - TFIIB complex:  N-terminal region of TFIIB forms Zn ribbon and B finger

**Clamp**

**Wall**

**IIB****N**

**Structure of pol II - TFIIB complex:  N-terminal region of TFIIB forms Zn ribbon and B finger**

**Saddle**

## Slide 36

## Slide 37

## Slide 38

## Slide 39

## Slide 40

## Slide 41

## Complete transcription bubble in TFIIF-2 complex

## Slide 43

## Slide 44

## H

**H**

**E**

**Pol**

**4/7**

**F**

**TBP**

**B****N**

**B****C**

## H

**H**

**E**

**Pol**

**4/7**

**F**

**TBP**

**B****N**

**B****C**

## H

**H**

**E**

**Pol**

**F**

**TBP**

**B****N**

**B****C**

## H

**H**

**E**

**Pol**

**F**

**TBP**

**B****N**

**B****C**

## H

**H**

**E**

**Pol**

**F**

**TBP**

**B****C**

## E

**E**

**Pol**

**F**

**TBP**

**B****C**

**H**

## Complete minimal RNA polymerase II 

**transcription initiation complex**

**Notes:**
This picture is assembled from both X-ray and electron crystallographic structures, as detailed in:  Bushnell, D.A., Westover, K.D., Davis, R., and Kornberg, R.D.  (2004)  Structural basis of transcription:  an RNA polymerase II – TFIIB cocrystal at 4.5 Å.  Science 303:983-988.

## Mechanism of initiation

**Role**

**Mechanism of initiation**
**of RNA polymerase II**
**transcription**

**H**

**Pol**

**TBP**

**B****C**

**H**

**Pol**

**TBP**

**B****C**

**F**

**B****N**

**Notes:**
See for details:   Bushnell, D.A., Westover, K.D., Davis, R., and Kornberg, R.D.  (2004)  Structural basis of transcription:  an RNA polymerase II – TFIIB cocrystal at 4.5 Å.  Science 303:983-988.

## Modular structure of Mediator

**Modular structure of Mediator**

## Head module crystals 

**proteins:  7**
**MW:  223 kDal**
**space group: C222**
**diffraction limit:  4 Å**
**a=236 Å, b=435 Å, c=328 Å**

## Slide 55