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Selected topics in Transcription

Selected topics in Transcription. Nir London. Computational Biology Seminar 2006. Overview. Elongation Pause; Arrest Chromatin remodeling; Histones CTD Mediator Complex Mechanism model Composition and Interaction network Initiation Mechanism New findings. Elongation.

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Selected topics in Transcription

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  1. Selected topics in Transcription Nir London. Computational Biology Seminar 2006

  2. Overview • Elongation • Pause; Arrest • Chromatin remodeling; Histones • CTD • Mediator Complex • Mechanism model • Composition and Interaction network • Initiation Mechanism • New findings

  3. Elongation • 17 BP Open bubble • 5’ to 3’ Elongation • 50-90 BP / second Leninger 5’th edition

  4. Elongation reaction • 3 ASPs highly conserved across all species Leninger 5’th edition

  5. Elongation by RNA polymerase II: the short and long of it Robert J. Sims, III, Rimma Belotserkovskaya and Danny Reinberg Genes & Dev. 2004

  6. What’s stopping elongation ? • Efficient elongation must overcome several blocks. • Transcriptional pause • Transcriptional arrest • Transcriptional termination • Many elongation factors serve to counteract or remove one of the above.

  7. Pause • The RNA polymerase halts elongation for a time before resuming on its own. • Pausing of bacterial RNA pol is caused by a structural rearrangement within the enzyme and DNA sequence.

  8. Easy modulation of rate ? • Demonstrated for all three eukaryotic RNA polymerases, viral and prokaryotic. • Pausing is self-reversible  a natural mode of transcriptional regulation. • Many factors modulate transcriptional pause and thus, the rate of elongation.

  9. Pause to cap • DSIF/NELF complex promotes pausing and enables capping • TFIIF < Elongins < ELLs promote elongation at different places along the gene.

  10. Arrest • Irreversible halt to synthesis. Pol cannot resume without additional factors • The polymerase “backtracking” relative to the DNA template • Misalignment of the catalytic site and 3-OH of the transcript • Pause decays into arrest in a time dependent fashion

  11. Resume mechanism • Resuming uses an evolutionarily conserved mechanism • Requires cleavage of the RNA transcript in a 3’-to-5’ direction • Cleavage allows the proper realignment of the active site and 3’-OH.

  12. TFIIS – Arrest solver • The cleavage reaction is intrinsic to the Pol. Enhanced in the presence of TFIIS.

  13. TFIIS (cont.) • An acidic hairpin coordinating a metal ion Re-aligns the RNA to the cleavage active site. Kettenberger H. et al. 2003

  14. Nucleosomes – another block • How does the Pol. Traverses the nucleosomes ? • Models: • Nucleosome mobilization • Histone depletion

  15. Swi\Snf – ATP dependent chromatin remodeler • Transcription pauses shortly after initiation. • HSF1 alleviates the negative effect of chromatin structure. • Recruits Swi\Snf to Hsp70 gene • Both Activator and Swi\Snf are required for transcription on nucleosomal templates.

  16. Mechanism ? Narlikar GJ. Et al. Cell 2002

  17. FACT – histone chaperone • Highly conserved • ChIP showed it to be localized downstream to promoters of active genes upon induction • Destabilize the nucleosome by removing one H2A/H2B dimer.

  18. Spt6 • Promotes nucleosome assembly in vitro • Spt6 mutants show alterations in chromatin structure • Colocalized to transcribed regions • Interacts with H3

  19. Mechanism

  20. Histone Modifications and elongation • Histone acetylation destabilizes chromatin structure • No evidence for a specific role of histone acetylation in elongation

  21. Set1/2 - Methylation • Methylation can co-map with silent or active regions – depend on Lys • Linking CTD to histone modifications • Set2 - H3-K36-specific histone methyltransferase • Set2 associates with the hyperphosphorylated RNAPII • Deletion of the CTD, or the CTD-kinase Ctk1, results in a loss of H3-K36 methylation • Set1 functions as a specific histone H3-K4 methyltransferase • Set1 interacts with the Ser-5 phosphorylated form of RNAP II. the form associated with early transcriptional events

  22. Elongator Chd1 Swi/Snf TFIIF Set1 Spt2 ISWII Set2 TFIIS DSIF Paf Iws1 Spt6 FACT P-TEFb

  23. CTD • CTD serves as a platform for many factors for mRNA maturation • Different phosphorylation patterns creates different structures

  24. Flexible • A) Cgt1-CTD • B) Pin1-CTD • Heptad repeats are not identical • Could explain specific factor binding

  25. Conclusions ? • Why are there so many redundant EF’s ? • The answer might be that they are promoter/gene specific • How does elongation and chromatin remodeling work together ? • How histone modifications translate to distinct functional outcomes ? • Why is the rate of elongation in vitro, far less than the rates observed in vivo ?

  26. The yeast Mediator complex andits regulation Stefan Bjorklund and Claes M. Gustafsson TRENDS in Biochemical Sciences, May 2005

  27. Mediator • Required for activator dependent stimulation of Pol2. • Comprised of 25 subunits • Can be found as free form or attached to Pol2.

  28. Mediator interaction with Pol2 • CTD reminder: • Initiation – unphosphorylated • Elongation – phosphorylated • Mediator complex interacts directly with the unphosphorylated form of the CTD • Dissociation upon elongation

  29. Transcriptional activation • The model: Mediator acts as a bridge between activators and basal Pol2 machinery.

  30. Transcription Transcription Transcription Transcription Activator Example – GAL4 • Gal4 interacts directly with subunits Med15, Med17. • ChIP showed association to be at an upstream activation sequence.

  31. 0 4-7 8-13 6-10 Mediator Pol II Galactose SAGA Separate recruitment • 3 waves of TF recruitment: • Separate recruitment has also been showed for other promoters. • Demonstrated in higher eukaryotes • Mediator forms a scaffold for several rounds of transcription

  32. Transcriptional repression • Srb8-11 identified as crucial for mediated repression • Tup1 repressor recruits Srb8-11 containing mediator • Srb10 kinase function is necessary for repression • Srb8-11 genes showed in genetic screens loss of repression

  33. Transcriptional repression • The model: repressors recruit mediator in a form in which interactions with Srb8-11 module are stabilized.

  34. Example – C/EBPβ • Switch phosphorylated by Ras • Active form recruits mediator devoid of Srb8-11 • Repressive form recruits Srb8-11 containing mediator

  35. Post translational modifications • Irregularities in SDSpage migration for certain subunits. • Treatment with phosphatase changed migration patterns • ATP-analog experiments showed that Kin28 (part of TFIIH) phosphorylates not only the CTD but also the mediator

  36. Modifications (cont.) • Other kinases target mediator: (Srb10, ras, PKA) • Another option for signaling pathways to modulate transcription • The effects of modifications aren’t characterized – Lots more to investigate

  37. Sub summary • Mediator influences both recruitment of Pol. and initiation of transcription • Might be involved in other transcription related processes (elongation, chromatin remodeling, splicing, RNA export) • How does PT modifications affect mediator function ?

  38. A high resolution protein interaction map ofthe yeast Mediator complex Benjamin Guglielmi, Nynke L. van Berkum, Benjamin Klapholz, Theo Bijma, Muriel Boube, Claire Boschiero, Henri-Marc Bourbon, Frank C. P. Holstege and Michel Werner Nucleic Acids Research, 2004.

  39. Strains expressing GBD-Med2, Med3, Med4, Med13, Med15 showed strong expression of b-gal and were excluded from this analysis. Pair-wise 2H analysis • Each subunit was cloned as fusion protein with Gal4 DNA binding domain (GBD) or Gal4 Activation domain (GAD). • Transformed into a GAL promoter-reporter genes strains. • All possible matings were preformed.

  40. Results

  41. Results (cont.) • Identified interactions were retested by co-transformation to same strain • 11 interactions found in middle-middle • 7 interactions in head-head • No interactions in tail

  42. Screening genomic lib. • Some interactions can’t be discovered using complete proteins • Same screen only now attached to GAD are random S. cerevisiae genomic seqs. • 17 interactions were found. (7 new ones)

  43. Med31 – new subunit • Med31 homologues found in mediator like complexes in higher eukaryots • Fusion with GBD against all other 24 showed 2 interactions in middle section • CoIP with Med17 confirmed it belongs to the mediator complex

  44. "בואו נחבוש את כובע הביקורת..."

  45. Interaction Domains Truncation of conserved areas reveals different interaction domains for Med subunits.

  46. Abortive Initiation and ProductiveInitiation by RNA PolymeraseInvolve DNA Scrunching Andrey Revyakin, Chenyu Liu, Richard H. Ebright, Terence R. Strick Science Nov. 2006

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