* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Document
Survey
Document related concepts
Cellular differentiation wikipedia , lookup
Hedgehog signaling pathway wikipedia , lookup
Signal transduction wikipedia , lookup
Biochemical switches in the cell cycle wikipedia , lookup
List of types of proteins wikipedia , lookup
Cell nucleus wikipedia , lookup
Histone acetylation and deacetylation wikipedia , lookup
Transcription factor wikipedia , lookup
Gene expression wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Eukaryotic transcription wikipedia , lookup
Transcript
Recap •eukaryotes have 3 nuclear RNA polymerases, which transcribe unique sets of genes •RNA pol II transcribes protein coding genes and must respond to and integrate a diverse set of signals in order to regulate expression of >25k genes •in vitro transcription systems for pol II show accurate initiation •gene specific regulators in euks have separable DNA binding and activation domains, the role of the DNA binding domain is to tether the activation domain near the promoter •activation domains have no clear distinguishing structural or sequence features that indicate their mechanism of action •squelching experiments indicate that activators compete for some limiting factor (not the polymerase) •TFIID and holoenzyme hypotheses may explain activator function activator interference or squelching activator B activator A UAS hypothesis? TATA box what is the limiting target of activators? 1. Eukaryotic activators do not bind to RNA pol II polymerase and therefore do not directly recruit polymerase to promoters. 2. Activators may, however, indirectly recruit RNA polymerase by recruiting factors (often called co-activators) that serve as a physical bridge between activator and polymerase. ‘TFIID hypothesis’ ‘Holoenzyme hypothesis’ Isolation of coactivators associated with the TATA-binding protein that mediate transcriptional activation Dynlacht, Hoey, Tjian, Cell 1991 QuickTime™ and a decompressor are needed to see this picture. Robert Tjian in vitro transcription reactions assembled from partially purified basal transcription factors -pol II ~90% pure -general factors <1% pure when assaying basal transcription (no activator present) in vitro, recombinant TBP can substitute for TFIID Isolation of coactivators associated with the TATA-binding protein that mediate transcriptional activation Dynlacht, Hoey, Tjian, Cell 1991 QuickTime™ and a decompressor are needed to see this picture. recombinant TBP cannot substitute for TFIID when assaying activated transcription in vitro Isolation of coactivators associated with the TATA-binding protein that mediate transcriptional activation Dynlacht, Hoey, Tjian, Cell 1991 TBP, the TATA-binding protein is small, but glycerol gradient sedimentation and gel filtration chromatography indicates that TFIID is very large Isolation of coactivators associated with the TATA-binding protein that mediate transcriptional activation Dynlacht, Hoey, Tjian, Cell 1991 the coactivator activity can be separated from TBP by ion exchange chromatography of TFIID in the presence of urea TBP and TBP-associated factors are required for activated transcription RNA Polymerase II Transcription Machinery Number of subunits Pol II 12 GTFs TFIID TFIIB TFIIE TFIIH TFIIF TFIIA* Mediator TBP TAFs * 1 12 1 2 9 2 3 22 Assembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriptional activators Chen et al., Cell 1994 had cloned and expressed most of the TAFs worked out methods for reconstitution of complex entirely from recombinant proteins Assembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriptional activators Chen et al., Cell 1994 Assembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriptional activators Chen et al., Cell 1994 TAFII150 and TAFII60 are sufficient for activation by NTF-1 Assembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriptional activators Chen et al., Cell 1994 NTF-1 activation domain peptide on beads TAFII150 and TAFII60 are specifically retained Assembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriptional activators Chen et al., Cell 1994 The ‘TFIID hypothesis’ QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. 1. TAFs provide surfaces for the interaction of TFIID with activators. QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. 2. TFIID recruits polymerase in vitro assays suggest specific activator-TAF contacts predictions? Yeast TAFII145 functions as a core promoter selectivity factor, not a general coactivator Walker et al., Cell, 1997 Shen and Green, Cell, 1997 polyA+ RNA levels are largely unaffected by inactivation of TAFs cell cycle regulated genes appear to be TAF-dependent Yeast TAFII145 functions as a core promoter selectivity factor, not a general coactivator Walker et al., Cell, 1997 Shen and Green, Cell, 1997 TAFII145 dependence tracks with the core promoter, not the UAS! Transcriptional activation via enhanced preinitiation complex assembly in a human cell-free system lacking TAFIIs Oelgeschlager et al., 1998 western blot demonstrating depletion of TAFIIs in vitro transcription shows that - transcription is abolished in the TFIID depleted extract - TBP is sufficient to restore activated transcription - 4 different activators were tested no transcription after depletion of TFIID and TAFs Conclusions: 1. Several activators can activate transcription in vitro in the absence of TAFs. 2. Not all transcription depends on TAFs in vivo. (based on analysis of yeast TAF mutants) 3. Some TAFs may assist in recognition of the core promoter (rather than transmitting regulatory information associated with upstream factors). 4. TAFs and alternative TBPs may specify selection of particular core promoters. What is the Limiting Target of Activators? activator B activator A UAS TATA box A novel mediator between activator proteins and the RNA polymerase II transcription apparatus (Kelleher et al. 1990) A novel mediator between activator proteins and the RNA polymerase II transcription apparatus (Kelleher et al. 1990) Gal4-VP16 TATA X UAS(dA-dT)2 UASGAL10 autoinhibition TATA activator interference UASGAL10 UAS(dA-dT)2 A novel mediator between activator proteins and the RNA polymerase II transcription apparatus (Kelleher et al. 1990) Gal4-VP16 Yeast nuclear extract in 50 mM (NH4)2SO4 X UAS(dA-dT)2 TATA 400 mM Elu QuickTi me™ and a TIFF ( LZW) decompressor are needed to see thi s pi ctur e. DEAE column 50 mM 50 mM FT 400 mM (NH4)2SO4 the 400 mM fraction overcomes squelching by Gal4-VP16 A novel mediator between activator proteins and the RNA polymerase II transcription apparatus (Kelleher et al. 1990) Potential explanations? -column fraction has activator for the template -something in column is binding/sequestering Gal4-VP16 -general stimulatory effect -fraction contains some limiting basal factor A novel mediator between activator proteins and the RNA polymerase II transcription apparatus (Kelleher et al. 1990) Potential explanations? -column fraction has activator for the template -no, it doesn’t squelch a Gal4 template -something in column is binding/sequestering Gal4-VP16 -no, activation by Gal4-VP16 is not disrupted -general stimulatory effect -no, activation depends upon Gal4-VP16 -fraction contains some limiting basal factor -no, adding them back does not overcome squelching Squelching in vitro: interpretation autoinhibition UASG TATA hypothetical target of activators UASG TATA excess Gal4-VP16 activator interference UASdA-dTTATA UASdA-dTTATA A mediator required for activation of RNA polymerase II transcription in vitro Flanagan et al., Nature, 1991 response to activators is lost during purification of general factors, but basal transcription (0ug Gal4-VP16) is unchanged mediator fraction restores activator response in a purified in vitro transcription system A mediator required for activation of RNA polymerase II transcription in vitro Flanagan et al., Nature, 1991 general transcription factors do not have mediator activity A mediator required for activation of RNA polymerase II transcription in vitro Flanagan et al., Nature, 1991 Gcn4 squelches Gal4-VP16 Gal4-VP16 squelches Gcn4 squelching is observed with the mediator fraction A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II (Kim et al. Cell, 1994) -Srb5 IP A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II (Kim et al. Cell, 1994) components of holo-RNA polymerase II: -12 polymerase subunits -3 TFIIF subunits -SRB proteins -Gal11, Sug1 RNA polymerase II Transcription Machinery Number of subunits Pol II 12 GTFs TFIID TFIIB TFIIE TFIIH TFIIF TFIIA* Mediator TBP TAFs * 1 12 1 2 9 2 3 22 Crystal Structure of Yeast RNA Polymerase II at 2.8 Å Resolution (Cramer et al, 2001) QuickTime™ and a YUV420 codec decompressor are needed to see this picture. CTD the carboxy-terminal domain (CTD) of RNA polymerase II QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. CTD facts: - unique to RNA pol II QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Heptapeptide of the CTD (52 repeats in mammalian Rpb1, 27 in yeast) - the CTD is not required for transcription in vitro -the CTD is essential for life -the CTD is subject to a cycle of phosphorylation at serines 2 and 5 - may be simultaneously phosphorylated at Ser2,5 A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II (Kim et al. Cell, 1994) an anti-CTD antibody separates mediator from RNA polymerase II An RNA polymerase II holoenzyme responsive to activators Koleske and Young, Nature, 1994 previously: •CTD truncation mutations limit the response to activators •isolated srb mutation as suppressors of CTD truncation mutations in yeast •purified a complex of Srb proteins with pol II and basal factors “RNA polymerase II holoenzyme” Srb proteins copurify with RNA pol II An RNA polymerase II holoenzyme responsive to activators Koleske and Young, Nature, 1994 previously: •CTD truncation mutations limit the response to activators •isolated srb mutation as suppressors of CTD truncation mutations in yeast •purified a complex of Srb proteins with pol II and basal factors “RNA polymerase II holoenzyme” Srb proteins copurify with RNA pol II holoenzyme supports activator dependent transcription in vitro Transcriptional activation via enhanced preinitiation complex assembly in a human cell-free system lacking TAFIIs Oelgeschlager et al., 1998 depletion of Srb7 decreased transcription response to activator The Med proteins of yeast and their function through the RNA polymerase II carboxy-terminal domain Myers et al., Genes & Dev., 1998 purified mediator binds the CTD in vitro The Med proteins of yeast and their function through the RNA polymerase II carboxy-terminal domain Myers et al., Genes & Dev., 1998 the CTD is required for mediator activated transcription in vitro a cycle of CTD modification during transcription TFIIH phosphorylates Ser5 at initiation P-TEFb phosphorylates Ser2 during elongation Ser5-P Ser2-P CTD phosphorylation: Pre-initiation initiation/escape elongation a cycle of CTD modification during transcription stage in transcription cycle pre-initiation early elongation phospho Ser 2, 5 later elongation CTD repeat=YS2PTS5PS the CTD phosphorylation cycle coordinates diverse events during transcription mediator stage in transcription cycle pre-initiation capping enzyme early elongation later elongation splicing and polyA factors Association of an activator with an RNA polymerase II holoenzyme Hengartner et al., 1995, Genes & Dev. holoenzyme is retained on a GST-VP16 column a mutation that abolishes activation by VP16 also abolishes holoenzyme binding Activation domain-mediator interactions promote transcription preinitiation complex assembly on promoter DNA Cantin et al., PNAS 2003 adenovirus E1A protein activates transcription of early genes by pol II E1A binds the Sur2 subunit of mediator in vitro and associates with mediator in vivo E1A mutations that prevent activation also disrupt Sur2 binding sur2-/- ES cells: -all other mediator subunits still in the complex -E1A doesn’t activate -several other activators still work Activation domain-mediator interactions promote transcription preinitiation complex assembly on promoter DNA Cantin et al., PNAS 2003 activation by E1A and Elk1 in vitro requires Sur2 no effect on activation by VP16 Activation domain-mediator interactions promote transcription preinitiation complex assembly on promoter DNA Cantin et al., PNAS 2003 Sur2 is required for binding of mediator and GTFs to E1A-bound promoters Nuc. extract 5x G4-act wash Holoenzyme Hypothesis Mediator serves as a physical bridge between RNA pol II and activators by which activators recruit polymerase to the promoter. Mediator Holoenyme TFIIE TFIIF Pol II TFIIH activator TBP TFIIB PIC Mediator Bound to RNA Polymerase II (Single Particle analysis) Clamp Tail F. Asturias Srb2 Srb4 Head Srb5 Head Srb6 Med6 Rgr1 Med8 Srb7 Med11 Med1 Middle Med4 Middle Med7 Rox3 Nut1 Sin4 Nut2 Med2 Tail Cse2 Med3 Gal11 2 conformations of mediator Holoenzyme Hypothesis Mediator serves as a physical bridge between RNA pol II and activators by which activators recruit polymerase to the promoter. Mediator Holoenyme TFIIE TFIIF Pol II TFIIH activator TBP TFIIB PIC mediator summary 1. Of the 20 mediator subunits in yeast, 13 had been identified previously in genetic screens for factors affecting transcription. 2. 11 mediator subunits are essential for life 3. Mediator appears to required for all pol II transcription (a general factor?) 4. Homologs for almost all Mediator subunits observed in fungi, plant and metazoan genomes. 5. Strong structural similarity observed between mediator complexes of yeast, mice, humans 6. In some cases, activators have been shown to contact specific mediator subunits and disruption of these contacts disrupts transcription Holoenzyme Hypothesis Mediator serves as a physical bridge between RNA pol II and activators by which activators recruit polymerase to the promoter. Mediator Holoenyme TFIIE TFIIF Pol II TFIIH activator TBP TFIIB PIC predictions of model? Gene activation by recruitment of the RNA polymerase II holoenzyme Farrel et al., Genes and Dev., 1996 recruitment of the mediator is sufficient for activated transcription QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Association of the Mediator complex with enhancers of active genes Kuras et al., PNAS 2003 Mediator Binding mediator binding did not depend on pol II binding or the TATA boxes TATA TATA GAL10 enhancer GAL1 The Swi5 activator recruits the Mediator complex to the HO promoter without RNA polymerase II Bhoite et al., Genes & Dev., 2001 Mediator can be recruited to genes independently of Pol II, GTFs and transcription -often, recruitment is to enhancers rather than core promoter Mediator as a general transcription factor Takagi and Kornberg, JBC, 2005 purified mediator from WT and srb4ts strains performed in vitro transcription reactions in the absence of activators “basal transcription” QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Mediator as a general transcription factor Takagi and Kornberg, JBC, 2005 1. mediator behaves like a general transcription factor 2. temp. shift experiment show that it is required prior to initiation QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Mediator as a general transcription factor Takagi and Kornberg, JBC, 2005 Srb4ts E(30°C) excess RNA pol II or basal factors cannot complement the transcription defect of the srb4ts mediator preparation suggests that mediator can act after recruitment of Pol II and general factors Some TAFs function in promoter recognition TFIIB TBP TAFs TATA TAFs Quick Time™ and a TIFF (LZW) dec ompressor are needed to s ee this pic ture. QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Inr DPE (Verrijzer et al, 1995) New Model: TAFs function in core promoter recognition Are all TAFs devoted to promoter recognition? QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Goodrich et al, (1996) TBP and TAF homologs may mediate tissue specific gene expression patterns in differentiated cells TRF=TBP related factor Reina JH, Hernandez N. Genes Dev. 2007