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Transcription. RNA Polymerases and General Transcription Factors. Subjects, covered in this lecture. A short overview Structure and mechanism of RNA polymerases Eukaryotic promoters General transcription factors Elongation factors

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

  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

  14. 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

  15. 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

  16. T7 The translocation in Pol II 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

  17. 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

  18. Substrate entry for T7 and RNA Pol II

  19. 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

  20. The eukaryotic promoters

  21. The TATA box

  22. 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 (TFIIBrecognition element) sequence, which has the consensus G/C-G/C-G/A-C-G-C-C, is locatedimmediately upstream of the TATA element of some promoters andincreases 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 startsite and includes a common G-A/T-C-G sequence

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

  24. 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 s factor is required

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

  26. The TATA box binding protein (TBP)

  27. The TATA box binding protein (TBP)

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

  29. The TATA box binding protein (TBP)

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

  31. Summary of interactions between TBP and TATA box Phe190 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) Phe99

  32. 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

  33. 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 NTD Pol II

  34. 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.

  35. 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

  36. TFIIE • TFIIE is a heterotetrameric protein (a2b2) • 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.

  37. 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

  38. Early events in elongation • 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

  39. 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.

  40. 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.

  41. The 3-D structure of Pol II holoenzyme

  42. 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

  43. TLFs as transcription regulators

  44. 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 Mg2+ • 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

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

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

  47. 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 thatregulate elongation of transcription (elongation factors) Elongation factors display following activities: • Enable elongation through chromatin • Suppress pausing • Overcome transcription arrest

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

  49. 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

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