In vitro systems have been used to investigate transcription.

1. In vitro systems have been used to investigate transcription. Transcription is reconstituted using: naked (histone-free) promoter DNA, purified RNA polymerase, NTPs, proteins purified from nuclear extracts (transcription factors). The RNA transcripts are analysed (quantity produced, start point). Mutagenesis is used to identify important bases within the promoter. Protein-binding sites within promoter DNA are mapped by footprinting.

2. Pol II promoters The minimal RNA pol II promoter has two key features. TATA box Initiator Inr TATA YYCAYYYYY -25 Start point

3. TATA – less promoters have a downstream positioning element (DPE) 28 to 32 nucleotides downstream of the start point. Initiator Inr DPE YYCAYYYYY AGAC + 30

4. Pol II initiation RNA pol II requires several transcription factors to initiate. TFIID is an 800 kDa protein complex. It consists of TATA box-binding protein (TBP) and eleven TBP-associated protein factors (TAFs).

5. TATA box-binding protein

6. TBP sits on the TATA box like a saddle.

7. TBP binding widens the minor groove and bends promoter DNA. Purified TBP protects the region from -37 to -25. Intact TFIID protects from - 45 to -10. Two of the TAFs contact the start point.

8. TFIID is the only basal transcription factor that makes sequence-specific contact with promoter DNA. Bound TFIID provides docking sites for other components of the transcription apparatus. The other TFIIs bind in a defined order: A, B, F + RNA pol II, E, H and J.

9. TFIIB binds the bent face of DNA and protects the region between -10 and + 10.

10. TFIID TFIIA TBP TFIIB TFIIA increases protection of the upstream region. This complex assists binding of Pol II + TFIIF

11. TFIID RNA pol II TFIIA TBP TFIIF TFIIB The initial complex of TFIID, TFIIA and TFIIB recruits RNA pol II to the promoter and positions it at the start point. Polymerase binding covers DNA as far as +15 on the template strand.

12. Then TFIIs E, H and J bind. TFIIH TFIIJ TFIID RNA pol II TFIIA TBP TFIIF TFIIB TFIIE TFIIE has helicase activity. TFIIH also has helicase activity and a kinase activity that phosphorylates the CTD of the largest pol II subunit.

13. Phosphorylation releases Pol II from the basal factors so that it can proceed to transcribe the template. Most of the TFIIs are released but TFIIH can reassociate. A form of TFIIH may function in DNA repair when RNA pol II is stalled by damage in the template. Some subunits of TFIIH are DNA repair proteins. Absence of one of these proteins (through mutation) results in the skin cancer Xeroderma pigmentosum.

14. Patients with Xeroderma pigmentosum

15. The TATA box and initiator are core elements that determine the start point and are sufficient for low level transcription in vitro. Upstream elements (UEs) are required for efficient transcription in vivo. UEs bind activator proteins that increase the efficiency of transcription initiation.

16. Upstream elements - are located between -40 and -110. - are generally quite short (~10 bp). - are orientation independent. - can have direct or inverted repeats (transcription factors may bind as dimers).

17. The CAAT box (consensus 5’ GGNCAATCT 3’) is recognised by NF1 transcriptional activators. The GC box (consensus 5’ GGGCGG 3’) is another common element that binds a 60 kDa activator protein Sp1. -110 - 40 -25 Start point CAAT box GC box TATA box

18. Activator bound to UE TFIID TFIID TATA Inr TATA Inr Factors bound to UEs contact basal factors TFIID, TFIIB or TFIIA. Protein-protein interactions favour assembly of thetranscriptioninitiation complex.

19. Promoters containing just core and upstream elements are active in all cell types. The number of UEs determines the basal level of transcription.