Biol 568 Advanced Topics in Molecular Genetics

1. Biol 568Advanced Topics in Molecular Genetics

2. Initiation of Transcription -Euk. General Overview Eukaryotic RNA Polymerases Transcription in Eukaryotes RNA pol II promoters Promoters / Enhancers

3. Overview Three eukaryotic RNA Polymerases: RNA Pol I transcribes rRNA RNA Pol II transcribes mRNA RNA Pol III transcribes tRNA & other

4. Overview RNA Pol II can�t initiate Txn alone need accessory factors General Factors Upstream Factors Inducible Factors

5. General Factors Required at all promoters Aid in determining txn start point Basal transcription factors

6. Upstream Factors Bind with specific short consensus sequences upstream of start site Activity not regulated Ubiquitous Required for promoters to function at adequate levels

7. Inducible Factors Similar to Upstream Factors Regulatory role Synthesized or activated as needed

8. Fig 21.1 Overview of control regions for a eukaryotic gene

9. Transcription Control Regions Promoter required for initiation generally located immediately upstream of start site Enhancer sequences that stimulate initiation may be located upstream or downstream of gene may be in either orientation

10. RNA Pol II Transcribes genes for mRNAs encoding proteins Highly sensitive to a-amanitin RNA Pol I not inhibited, RNA Pol III variable inhibition (depending on source) Large protein - 500kDal - 8-14 subunits

11. RNA Pol II Purified enzyme can transcribe Can not initiate selectively at promoters

12. Fig 21.2 Yeast RNA Pol II subunits

13. Promoter Analysis Defined by ability to initiate transcription in test system Define the overall length Define the cis-acting element Once promoters are defined, the interacting proteins can be identified

14. Promoter Analysis Three test systems: Oocyte system Transfection systems Transgenic systems

15. Oocyte system Injection of DNA template into nucleus of Xenopus laevis oocyte RNA recovered and analyzed Limited to conditions in X.laevis oocyte Can be used for characterization of promoter sequences The specific interacting proteins may not be identified

16. Transfection Systems Exogenous DNA is introduced into cultured cells and expressed Conditions similar to in vivo Can be done with more than one gene Cotransfection

17. Transgenic Systems Addition of gene to germline of animal �Transgene� expression can be followed in any tissue of animal Gene may be present in multiple copies Gene may have integrated at different chromosomal location

18. Fig 21.3 Promoter Analysis (deletion)

19. Definition of promoter boundaries

20. Promoter Functional Analyses Deletion analyses Mutation analyses (bp changes) Protein footprinting Promoter sequence comparison Look for consensus sequence

21. Initiation of Transcription -Euk. General Overview Eukaryotic RNA Polymerases Transcription in Eukaryotes RNA pol II promoters Promoters / Enhancers

22. RNA Pol II promoter Initiator (Inr) First base generally an �A� Flanked by pyrimidines C/T; �Y�s Y2CAY5 (-3 to +5)

23. RNA Pol II promoter TATA box usually at -25 found in all eukaryotes 8bp consensus sequence generally all A/T analogous to bacterial -10 sequence

24. RNA Pol II promoter TATA box Mutations; �down� mutations Surrounded by GC sequences, function not known TATA-less promoters DPE; Downstream promoter element 28-32 bp downstream of the start point

25. Fig. 21.10 RNA pol II promoter Initiatior region; InR TATA + InR InR + DPE

26. RNA Pol II basal apparatus RNA Pol II can�t initiate txn itself Additional protein factors required Transcription factors For RNA Pol II - TFII X X identifies the individual factor

27. RNA Pol II Initiation First factor to bind to promoter is TF II D binds at TATA box contains TATA binding protein subunit (TBP) other subunits are TAFs -TBP associated factors TAFs may differ, and thus confer specificity TAFII43, TAFII230, etc.

28. Fig 21.11 Positioning RNA Pol II

29. Fig 20.9 TBP bound to DNA

30. Fig 21.13 TBP bound to DNA

31. TBP binds to DNA Protected DNA region ( in vitro) -37 to -25, ~ 1 turn TAFII230 competes for TBP DNA binding region

32. Add discussion about histone interactions? Some TFs have histone-like structures

33. Fig 21.15 Additional TFIIs bind

34. Fig 20.12 TF II B Interactions

35. Fig 20.12 TF II B Interactions

37. TFIIB interacts with RNA pol II

38. Fig 21.15 Additional TFIIs bind

39. TFIIF Two subunits RAP74; helicase activity RAP38; similarities with s factors Binds tightly to RNA pol II TFIIF may bring RNA pol II to the complex

40. RNA pol II holocomplex TF II D TBP binds and bends DNA TF II A Binds upstream of TBB TF II B Binds downstream and upstream of TATA TF II F and RNA Pol II RNA pol II interacts with TBP and TAFs via CTD RNA pol II interacts with TF IIB TF IIF melts DNA

41. TATA-less promoters Inr, DPE binding elements for specific TAFs Same general transcription factors TFIID TBP Bending and binding maybe carried out by specific TAFs

42. Comparison with txn in prokaryotes Bacterial RNA pol Intrinsic DNA binding activity s factors provide specificity First form complex with the core enzyme RNA pol II Cannot bind to DNA Txn factors provide specificity First bind to DNA then form complex with RNA pol II Independent s factors

43. TFIID TFIIA TFIIB TFIIF and RNA Pol II TFIIE TFIIH & J

44. Txn; Promoter Clearance TFIIE Helicase activity TFIIH & J Helicase activity Kinase activity

45. Fig 21.17 Release of RNA Pol II from promoter

46. Release of RNA Pol II from promoter Most of TFs are released prior to RNA Pol II continuing elongation CTD tail is phosphorylated by TFII H and other factors (YSPTSPS)

47. Phosphorylation of CTD CTD is involved in post-txn modifications Capping Guanylyl transferase binds to phosphorylated CTD Splicing SCAFs bind to CTD, may in turn bind to splicing proteins Phosphorylation of CTD may also be a mean to coordinate txn and processing

48. Initiation of Transcription -Euk. General Overview Eukaryotic RNA Polymerases Transcription in Eukaryotes RNA pol II promoters Promoters / Enhancers

49. Promoter Analysis Start site TATA box Short sequence elements upstream of TATA box Recognized by activators Provide txn efficiency and specificity

50. Fig 21.21 Saturation mutagenesis of a b-globin promoter

51. Modularity of Eukaryotic Promoters Short sequence elements are functional Binding sites for TFs Mix and match of elements provides level of control needed

52. Modularity of Eukaryotic Promoters Type of element Number of elements Position of elements Determine whether a gene is expressed and level of expression

53. Fig 21.22 Promoters contain different combinations of elements

54. Element - TF interactions GC box factor SP1 binds upstream factor position and number vary in promoters

55. Element - TF interactions CAAT box upstream factor recognized by several TFs CP1, CP2 (CTF family) C/EBP ACF boxes in different promoters recognized by different factors

56. Element - TF interactions Octamer element upstream element bound by two TF Oct-1 ubiquitous TF (non-lymphoid cells) Oct-2 only lymphoid cells (activates k-light gene)

57. Element - TF interactions Context of elements must be important Oct 1 doesn�t activate k-light genes in non-lymphoid cells Presence of specific elements does not mean that the specific TF activates txn May need to interact with other TFs Protein - protein interactions

58. Role of Enhancers Boost level of transcription Act in conjunction with promoter Cis- acting elements Position relative to promoter is not fixed upstream or downstream Can work in either orientation

59. Fig 20.19 Enhancer mutational analysis

60. Enhancers Modular nature, like promoters Higher density of TF binding sites Some elements found in both enhancers and promoters Increase Txn up to 200 fold!

61. What is the function of the enhancer? Brings transcription factors close to the promoter DNA looping May initiate chromatin remodeling Insulators may inhibit an enhancer function

62. Initiation of Transcription -Euk. General Overview Eukaryotic RNA Polymerases Transcription in Eukaryotes RNA pol II promoters Promoters / Enhancers