General  Transcrip-on  Factors:  TBP/TFIID    TBP  Associated  Factors:  TAFs    (Elonga-on  of  Transcrip-on)  

h?p://www.fmi.ch/Training/Teaching/  

Transcrip)on  Regula)on  And  Gene  Expression  in  Eukaryotes    

Cycle  G2  (lecture  13709)  FS  2014  P  Ma?hias  &  RG  Clerc    

 P.  Ma?hias,  March  5th,  2014  

 The  Basics  of  Transcrip-on  (2)

Factors needed for expression of nearly all Class II promoters Biochemical Fractionation (R. Roeder, Rockefeller University) led to identification of 6 fractions that contain proteins that permit correct transcription initiation by RNA pol II These fractions were labeled TF II: A, B, D, E, F, H. (Fraction C was an artefact.) Of these TFIID, TFIIA and TFIIB bind specific proximal elements (others such as TFIIH bind DNA but are not thought to have strict DNA sequence preference) TFIID nucleates the assembly of the pre-initation complex (PIC)

GTFs: recognize the core promoter

TATA INR

core

GENE

upstream (regulatory, enhancer)

Basal machinery

RNA PolII 12 TFIIA 3 TFIIB 1 TFIID (TBP) ca. 14 (1) TFIIE 2 TFIIF 2 TFIIH ca. 9

Transcription activators NF-kB

Fos Oct-1 CBFA-1 CREB etc…

Eukaryotic Pol II promoter: upstream and core

TFIIB!RNAPII!

TFIIA!TFIID!

TFIIH! TFIIF!TFIIE!

PyPyANT/APyPy GA/TCGTG TATAAA G/CG/CG/CCGCC

DPE Initiator TATA box BRE

+31 -32 -26 +1

The core promoters in more detail

TAFII250 TAFII150

TAFII60 TAFII40

TBP

TFIIB TFIID TFIID TFIID

TFIIA

TATA-less

TATA-containing

TFIID  

•  TFIID  is  a  mul-protein  complex:  TBP  +  TAFs  –  (~10-­‐14  different  subunits)  

•  The  subunit  responsible  for  recognizing  the  TATA  box  is  called  ‘TATA-­‐Binding  Protein’  (‘TBP’)  

•  TBP  can  specifically  bind  to  TATA-­‐boxes  on  its  own  

•  In  vitro  TBP  is  sufficient  for  “basal”  transcrip-on,  not  for  “ac-vated”  transcrip-on  

Akhtar  and  Veenstra  Cell  &  Bioscience  2011,  1:23    h?p://www.cellandbioscience.com/content/1/1/23      

TBP: a GTF essential for all polymerases

TAFIIs of TFIID: mostly conserved proteins ranging from 30 à 250 kDa

TBP-related factors

Akhtar  and  Veenstra  Cell  &  Bioscience  2011,  1:23    h?p://www.cellandbioscience.com/content/1/1/23      

Akhtar  and  Veenstra  Cell  &  Bioscience  2011,  1:23    h?p://www.cellandbioscience.com/content/1/1/23      

TBP-related factors

TBP-related factors specific complexes

Promoters with TBP and TRFs

Goodrich  &  Tjian,  NRG  Aug  2010      

Albright and Tjian (2000). TAFs revisited: more data reveal new twists and confirm old ideas. Gene 242, 1-13.

Albright and Tjian (2000). TAFs revisited: more data reveal new twists and confirm old ideas. Gene 242, 1-13.

Beyond TFIID: Multiple TAF complexes

Beyond TFIID: Multiple TAF complexes

TFIID!TBP! }!TAFs!IIB!

IIA!IIE!

IIF!IIH!

helicase!

protein kinase!

TBP!Inr!IIB! IIA! Pol IIa!

IIF!IIE!IIH!

CTD of large subunit of Pol II!

Recognize core promoter!Targets Pol II !to promoter!

Modulates helicase!

Helicase!

CTD protein kinase!

Summary

Cell  and  stage-­‐specific  dynamics  in  basal  factors  

D’  Alessio  et  al.,  Molecular  Cell  36,  December  24,  2009      

Goodrich  &  Tjian,  NRG  Aug  2010      

Goodrich  &  Tjian,  NRG  Aug  2010      

Goodrich  &  Tjian,  NRG  Aug  2010      

Gene  bookmarking  during  mitosis  

TATA  vs  TATA-­‐less  promoters  

S  Buratowski,  Nature  Vol  483,  2012  (News  &  Views)  See  Rhee  &  Pugh,  Nature  Vol  483,  2012    

TATA-­‐less  promoters:  have  TATA-­‐like  elements  

6000  PICs  localized  in  yeast  

A  short  story  ca.  15-­‐20  years  ago:  What  are  the  in  vivo  targets  of  transcrip-on  ac-vators???  

East  coast  vs  West  coast,  Yeast  vs  Metazoans,  In  vivo  vs  in  vitro,  ...  

GTFs TBP

Sp1 + TFIID

+

TBP vs TFIID: TAFs as universal coactivators??

In  vitro  txn  assay  

Which part of the promoter is TAF responsive?

In vivo rates of transcription = 1200-2000 nucl/min. Baseline in vitro rates = 100-300 nucleotides/min.

frequent pausing and arrests even with chromatin-free DNA Large number of proteins and protein complexes that

regulate elongation of transcription • Suppress pausing • Promote pausing • Overcome transcription arrest • Enable elongation through chromatin

Transcription Elongation

• Suppress pausing and/or arrest TFIIF P-TEFb (CTD kinase) S II (TFIIS) CSB (Cockayne syndrome binding pro.) ELL 1/2, Elongins certain transcription activators (e.g. heat shock factor)

• Promotes pausing/arrest DSIF (DRB sensitivity inducing factor) NELF (binds early during RNA elongation)

• Enable elongation through chromatin FACT RSF Elongator (HAT associated with Pol II)

Elongation factors for RNA Pol II

Elongation

NMCB,  August  2006  

Elongation