1. Transcription RNA Polymerases and General Transcription Factors

2. Subjects, covered in this lecture A short overview Structure and mechanism of RNA polymerases Eukaryotic promoters General transcription factors Elongation factors Shortly about eukaryotic RNA polymerases, other than RNA Pol II

3. Nucleic acid polymerases: classification Nucleic acid polymerases RNA polymerases DNA polymerases DNA dependent DNA polymerases DNA dependent RNA polymerases RNA dependent DNA polymerases RNA dependent RNA polymerases Template specificity HIV reverse transcriptase HCV replicase E.coli DNA polymerase III RNA polymerase II Sugar specificity Adds dNTPs Adds rNTPs

4. Sugar specificity – how? dNTP- yes! ; rNTP – no!  that’s easy! some residue blocks entry of 2’OH

5. Sugar specificity – how? rNTP- yes! ; dNTP – no!  not easy!

6. Simple and complex DNA – dependent RNA polymerases Viral RNA polymerases (1 subunit) Prokaryotic RNA polymerases (4 different subunits) Eukaryotic and archeal RNA polymerases (at least 12 subunits) Viruses

7. The structural features of RNA polymerases Based on 3D sructures of bacteriophage T7 RNA pol and yeast RNA Pol II

8. The bacteriophage T7 RNA polymerase: structure and mechanism

9. The transcription bubble To solve the structure, transcription bubble oligos and ATP analogues were added to crystallization mixture Methylene group, to prevent the product formation No OH group, to prevent addition of the next nucleotide

10. The catalytic reaction mechanism of T7 RNA polymerase

11. Active site in 3D

12. Pre- And Post-translocation structures After release of PPi the O helix looses contact with active site asparagines and gets rotated. The rotation displaces Tyr639, which in turn moves the RNA, resulting in a translocation event Tyr 639 has three functions –(1) sugar discrimination, (2) stacking against incoming base and (3) translocation

13. The nucleotide addition cycle

15. Differences between non-transcribing and transcribing structures of RNA Pol II The non-transcribing complex is more “open”, which faciliates DNA binding Upon the binding to promoter, the structure, called “clamp” closes the complex like a lid

16. Maintenance of transcription bubble Lid, Rudder, Zipper – separates DNA from RNA Fork loops – separates incoming DNA strands Bridge helix – translocates the bubble Wall – holds the hybrid in position Pore 1 – nucleotide income

17. The translocation in Pol II The bridge helix is thought to change the conformation from straight to bent, resulting in the translocation event, somewhat similarly as in T7 RNA polymerase However, bridge helix interacts with DNA, not RNA as in T7 case. Also, unlike T7, there is no tyrosine involved in translocation T7 Pol II

18. Are multi-subunit (RNA pol II) and single subunit (T7) RNA polymerases evolutionary related? Probably not One similar feature is involvement of two magnesium ions in catalytic activity Another similar feature is involvment of Bridge helix (Pol II) and O helix (T7) in translocation, although there is no involvement of tyrosine in Pol II However, there are no sequence or structure similarities among T7 and Pol II Differences exist also in ribose recognition – no obvious sugar discriminators in PolII

19. Substrate entry for T7 and RNA Pol II

20. Carboxyl terminal domain (CTD) of Pol II A stretch of 7aa repeats in c-terminus of Pol II subunit 1. Consensus sequence of repeats YSPTSPS In yeast 26 repeats, in humans - 50 CTDs get phosphorylated at several Ser and Tyr residues after transcription initiation Phosphorylation of CTDs is important for promoter escape, elongation and post-transcritption events CTDs have shown to act as a “landing pad” for different proteins, involved in elongation and post-transcription events The base of CTDs is located near the exit groove of RNA

21. The eukaryotic promoters

22. The TATA box

23. Other promoter sequences Initiator (Inr): Y-Y-A +1 -N-T/A-Y-Y-Y. Some promoters contain both initiator and TATA box, whereas others only one of them CpG islands: CG rich stretch of 20-50 nucleotides within ~100 base pairs upstream the start site. BRE (TFIIB recognition element) sequence, which has the consensus G/C-G/C-G/A-C-G-C-C, is located immediately upstream of the TATA element of some promoters and increases the affinity of TFIIB for the promoter Downstream promoter element (DPE) is present in some genes with initiator promoter. It is located about 30 nucleotides downstream of the transcription start site and includes a common G-A/T-C-G sequence

24. Summary of promoter elements CpG island approx. -100 to -1

25. General transcription factors General transcription factors are required for transcription in eukaryotes from all genes GTFs assist RNA Pol II in transcription initiation GTFs are designated TFIIA, TFIIB,... and most of them are multimeric proteins Equivalent GTFs are highly conserved among the eukaryotes In prokaryotes, only one general transcription factor, known as  factor is required

26. TFIID TFIID is composed of 14 subunits, one of them being TATA box binding protein, TBP Functions: promoter recognition, TFIIB recruitment

27. The TATA box binding protein (TBP)

29. Stacked bases Stacking between Phe and A base, NOT between 2 bases

30. The TATA box binding protein (TBP)

31. Steric hindrance if A was G Val Thr Asn A T TATATAAA ATATATTT

32. Summary of interactions between TBP and TATA box TATA box is making a pseudo- twofold sequence-specific interaction with two threonines and two asparagines of TBP Stacking of phenylalanines against DNA bases is also shown (DNA bending due to stacking is not shown) Phe190 Phe99

33. TFIIB Functions: - start site selection for Pol II - TFIIF-PolII complex recruitment Some TFIIB mutants result in a shift of transcription start site Under certain conditions (BRE element promoter) Pol II together with TFIID and TFIIB can form the minimal initiation complex. At most promoters, however, TFIIE, F and H are necessary

34. C-terminal domain (CTD) of TFIIB CTD of TFIIB interacts with both TBP and DNA around the promoter – especially BRE element Rough positioning of Pol II is due to interaction of TFIIB CTD with TBP Fine positioning is due to interaction with DNA N-terminal domain of TFIIB interacts with Pol II Pol II NTD

35. TFIIF Functions: - Recruitment of Pol II to the existing DNA-TFIID-B complex, - Positioning Pol II over the start site - Binding to the non-template DNA strand. - TFIIF also reduces non-specific binding of RNA pol II to DNA.

36. The 3-D structure of TFIIF TFIIF is a heterotetramer, made from two subunits of RAP30 and two subunits of RAP74 proteins RAP30-RAP74 dimer within the complex structure has an unusual triple-barrel fold

37. TFIIE TFIIE is a heterotetrameric protein ( a 2 b 2 ) Functions: - TFIIE appears to create the docking site for next transcription factor, TFIIH. - TFIIE also modulates TFIIH enzymatic activities - In addition TFIIE enhances promoter melting.

38. TFIIH TFIIH is a multimeric protein, composed of 9 subunits, some of them with distinct enzymatic activities Functions: - TFIIH has a helicase activity, which unwinds the DNA duplex at a start site, allowing Pol II to bind to the template strand. - TFIIH also has a kinase ativity, it phosphorylates PolII in the begining of elongation - Other TFIIH subunits have been shown to recruit DNA-repairing enzymes if polymerase reaches damaged region in DNA and gets stalled

39. As Pol II transcribes away from the start site subunit of TFIIH phosphorylates the Pol II CTD, which results in promoter escape. General factors get released Early events in elongation

40. TFIIA For transcription in vivo, another factor TFIIA is required The function of TFIIA is somewhat unclear, but it might help the other factors to bind. TFIIA has also shown to have some anti-repressor functions TFIIA is not required for transcription in vitro.

41. TAFs Apart from TBP, TFIID has 13 TAF ( TBP associated factors) subunits Some of them seem to be necessary for transcription initiation from promoters, lacking the TATA box Other TAFs have been shown to be tissue- specific coactivators TAF subunits also interact with other GTFs therefore stabilyzing the complex.

42. The 3-D structure of Pol II holoenzyme

43. TBP-like proteins (TLFs) TBP-like proteins are TBP protein analogues (not to be confused with TAFs) Some TLFs reconize TATA box Other TLFs recognize some differnt promoter sequence TLFs are thouht to play a role in transcription regulation

44. TLFs as transcription regulators

45. What is Pol II backsliding, pausing and arrest? Backsliding is an event when RNA polymerase moves backwards and newly made RNA gets inserted in the funnel. It can be caused by incorporation of wrong NTP or when 3’OH looses contact with active site Mg 2+ If the backsliding RNA piece is 2-4 nt long, this a reversible process and RNA polymerase can recover by itself. This is called pausing. If the backsliding RNA gets longer (7-10 nt), it gets trapped in funnel pore and RNA polymerase can not recover by itself. This is caused arrest. Arrest can be overcome by specific elongation factors, which help RNA Pol II to cleave the arrested RNA

46. Backsliding RNA Backsliding can be caused, when 3’ hydroxyl looses contact with Mg

47. Movement of RNA and DNA in transcription and backsliding Movement of RNA and DNA is indicated with arrows Transcription Backsliding

48. Transcription elongation factors In vivo rates of transcription = 1200-2000 nucleotides/min. Baseline in vitro rates = 100-300 nucleotides/min. In vitro frequent transcription pausing and arrests occur even with chromatin-free DNA Large number of proteins and protein complexes exist that regulate elongation of transcription (elongation factors) Elongation factors display following activities: Enable elongation through chromatin Suppress pausing Overcome transcription arrest

49. Transcription elongation factor IIS Elongated shape –why? TFIIS gets inserted in active site of PolII

50. The 3D structure of TFIIS-Pol II complex Domain III of TFIIS gets inserted in the active site of PolII Domain II docks on the surface

51. Interaction of TFIIS acidic hairpin with arrested RNA THe acidic residues D290 and E291 assist in cleaving the phosphodiester bond The backtracked RNA fragment gets released and RNA Pol II is rescued from arrest

52. Elongator decondenses the chromatin Sequential histone acetylation by transcription-coupled histone acetyltransferase (part of elongator complex)

53. The ebb and flow of histones The histone chaperone activity of Spt6 helps to redeposit histones on the DNA thus resetting chromatin structure after passage of the large RNAPII complex

54. Other elongation factors P-TEFb (Positive Transcription Elongation Factor) phosphorylates additional residues in CTD of PolII (some are already phosphorylated by TFIIH). ELL family of proteins also stimulates transcription elongation through a poorly understood mechanism. There is some evidence, that ELLs target RNA polymerase for degradation upon reaching damaged regions in DNA. Degradation of PolII is followed by recruitment of DNA repairing enzymes. Negative factors NELF and DSIF supress elongation by direct interaction with Pol II

55. Other eukaryotic transcription systems RNA pol I (pre-rRNA) RNA pol III (tRNA, 5S rRNA) Mitochondrial RNA polymerase Chloroplast RNA polymerase

56. RNA Pol I

57. RNA pol III Unlike other polymerases, promoter sequences lie entirely within coding sequence. The conserved A and B sites also code for two invariant sequences, observed in all tRNAs Three initiation factors are reqired for Pol III initiation. TFIIIA is required only for 5S rRNA transcription. TFIIIB has three subunits, one of them similar to TFIIB and another one being TBP (same as for Pol I and II)

58. Mitochondrial RNA polymerase Encoded in nuclear RNA and transported to mitochondrial matrix Contains only two subunits One subunit similar to bacteriophage T7 RNA polymerase Other subunit similar to bacterial s factor s -like s b b’b’ a a w T7-like

59. Chloroplast RNA polymerase Encoded by chloroplast genome Contains considerable homology to bacterial ,  and  ’ RNA pol subunits No any s -like factors or general transcription factors s b b’b’ a ab b’b’ a a w