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In vitro assembled pre-initiation complex
• TFII yeast fly/man• A 2 3• B 1 1• D 1 (15 TAFs) 1 (14/15 TAFs)• E 2 2• F 3 2• H 9 1• Pol II 12 12• Total-TAFs 30 29• Total+TAFs 45 43/44
Do such assemblies actually form at a promoter?
• The assembly time is very long in vitro relative to normal rates of transcription initiation in vivo
• The in vitro assemblies are on DNA not an in vivo template (chromatin)
• The holoenzyme makes more sense
Yeast RNA Polymerase II Holoenzyme.
The CTD in the elongating complex
What are SRBs?• SRB genes were identified in a genetic screen in yeast
as dominant gain of function suppressors of CTD truncation hence: suppressor of RPB1
• The wild type RPB1 was replaced by a CTD deletion mutant which was conditionally lethal
• Mutations in the SRB genes; 4, 5, and 6 allowed normal growth
• It is thought that SRBs function to allow activators to work with the deleted CTD
SRBs exist in various complexes
• The holoenzyme has SRBs; 2, 4, 5, 6, 7, 8, 9, 10 and 11
• SRBs 2, 4, 5, 6 exist as an isolated complex• SRBs 2, 4, 5, 6, 7 are associated with
components of the mediator complex in another isolated complex
SRBs 10 and 11
• SRB 10 is cyclin C• SRB 11 is cdk8• Together they can phosphorylate the CTD in
addition to TFIIH’s cyclin H and cdk7 at S5
What is the mediator?• The activity was identified by its ability to stimulate
specific transcription from a purified pol II system 10-fold
• The mediator also stimulates activator response in the same pol II system 30-fold
• In combination this results in a 300-fold activation of basal transcription
• The mediator also stimulates CTD phosphorylation by TFIIH 30-50 fold
Mediator and Holoenzyme
MediatorStructure
MediatorbindstoAc1vatorboundtoDNAthenassociateswithPIC
Mediator-PolII-Pre-ini1a1onComplex
CTD Kinases and Elongation
Activators are modular
• DNA binding domain• Transcriptional activation domain• Oligomerization domain• Other specificity or protein-protein interacting
domains
12-18
DNA-BindingDomains
• Proteindomainisanindependentlyfoldedregionofaprotein
• DNA-bindingdomainshaveDNA-bindingmo1f– Partofthedomainhavingcharacteris1cshapespecializedforspecificDNAbinding
– MostDNA-bindingmo1fsfallinto3classes;zinc-containingmodules,homeodomainsandbZIPandbHLHmo1fs
12-19
12.2StructuresoftheDNA-BindingMo1fsofAc1vators
• DNA-bindingdomainshavewell-definedstructures
• X-raycrystallographicstudieshaveshownhowthesestructuresinteractwiththeirDNAtargets
• Interac1ondomainsformingdimers,ortetramers,havealsobeendescribed
• MostclassesofDNA-bindingproteinscan’tbindDNAinmonomerform
12-20
Zinc-ContainingModules
• Thereareatleast3kindsofzinc-containingmodulesthatactasDNA-bindingmo1fs
• Alluseoneormorezincionstocreateashapetofitanα-helixofthemo1fintotheDNAmajorgroove– Zincfingers– Zincmodules– Modulescontaining2zincand6cysteines
12-21
ZincFingers
• DescribedbyKluginGTFTFIIIA• Ninerepeatsofa30-residueelement:
– 2closelyspacedcysteinesfollowed12aminoacidslaterby2closelyspacedhis1dines
– Coordina1onofaminoacidstothemetalhelpsformthefinger-shapedstructure
– Richinzinc,enoughfor1zincionperrepeat– Specificrecogni1onbetweenthezincfingeranditsDNAtargetoccursinthemajorgroove
Zn Finger.
A-helix b-pleated sheet.
Znfinger1ofZif268rightsideisaan1parallelβ-sheetontherightsideisanα-helix
ArrangementofthreeZnfingersofZif268
Three fingers binding to DNA.
12-26
TheGAL4Protein
• TheGAL4proteinisamemberofthezinc-containingfamilyofDNA-bindingproteins
• EachGAL4monomercontainsaDNA-bindingmo1fwith:– 6cysteinesthatcoordinate2zincionsinabimetalthiolatecluster
– Shortα-helixthatprotrudesintotheDNAmajorgrooveistherecogni1onmodule
– Dimeriza1onmo1fwithanα-helixthatformsaparallelcoiledcoilasitinteractswiththeα-helixonanotherGAL4monomer
Gal 4 binding to DNA.
12-28
TheNuclearReceptors
• Athirdclassofzincmoduleisthenuclearreceptor
• Thistypeofproteininteractswithavarietyofendocrine-signalingmolecules
• Proteinplusendocrinemoleculeformsacomplexthatfunc1onsasanac1vatorbybindingtohormoneresponseelementsands1mula1ngtranscrip1onofassociatedgenes
12-29
TypeINuclearReceptors
• Thesereceptorsresideinthecytoplasmboundtoanotherprotein
• Whenreceptorsbindtotheirhormoneligands:– Releasetheircytoplasmicproteinpartners– Movetonucleus– Bindtoenhancers– Actasac1vators
12-30
Glucocor1coidReceptors
• DNA-bindingdomainwith2zinc-containingmodules
• OnemodulehasmostDNA-bindingresidues
• Othermodulehasthesurfaceforprotein-proteininterac1ontoformdimers
Glucocor1coidreceptorDNArecogni1onhelix
12-32
TypesIIandIIINuclearReceptors
• TypeIInuclearreceptorsstaywithinthenucleusboundtotargetDNAsites
• Withoutligandsthereceptorsrepressgeneac1vity
• Whenreceptorsbindligands,theyac1vatetranscrip1on
• TypeIIIreceptorsare“orphan”whoseligandsarenotyetiden1fied
TheAntennapediaphenotype-homeo1cgenes
12-34
Homeodomain-DNAComplex• HomeodomainscontainDNA-bindingmo1ffunc1oningashelix-turn-helixmo1fs
• Arecogni1onhelixfitsintotheDNAmajorgrooveandmakesspecificcontactsthere
• N-terminalarmnestlesintheadjacentminorgroove
12-35
ThebZIPandbHLHDomains
• bZIPproteinsdimerizethroughaleucinezipper– Thisputstheadjacentbasicregionsofeachmonomerinposi1ontoembraceDNAtargetlikeapairoftongs
• bHLHproteinsdimerizethroughahelix-loop-helixmo1f– AllowsbasicpartsofeachlonghelixtograsptheDNAtargetsite
• bHLHandbHLH-ZIPdomainsbindtoDNAinthesameway,theformerhaveextradimeriza1onpoten1alduetotheirleucinezippers
Leucinezipperisaparallelcoiledcoil
Leucine zipper bound to DNA.
bZipmo1fofGCN4boundtoitsDNATarget
Leucine zippers.
bHLH Myo D bound to DNA.
StructureofthebHLHdomainofMyoDboundtoitsDNAtarget
12-40
12.3IndependenceoftheDomainsofAc1vators
• DNA-bindingandtranscrip1on-ac1va1ngdomainsofac1vatorproteinsareindependentmodules
• MakinghybridproteinswithDNA-bindingdomainofoneprotein,transcrip1on-ac1va1ngdomainofanother
• Thehybridproteins1llfunc1onsasanac1vator
12-41
12.4Func1onsofAc1vators
• BacterialcoreRNApolymeraseisincapableofini1a1ngmeaningfultranscrip1on
• RNApolymeraseholoenzymecancatalyzebasalleveltranscrip1on– O`eninsufficientatweakpromoters– Cellshaveac1vatorstoboostbasaltranscrip1ontohigherlevelinaprocesscalledrecruitment
12-42
Eukaryo1cAc1vators• Eukaryo1cac1vatorsalsorecruitRNApolymerasetopromoters
• S1mulatebindingofgeneraltranscrip1onfactorsandRNApolymerasetoapromoter
• 2hypothesesforrecruitment:– GeneralTFcauseastepwisebuild-upofpreini1a1oncomplex
– GeneralTFandotherproteinsarealreadyboundtopolymeraseinacomplexcalledRNApolymeraseholoenzyme
12-43
ModelsforRecruitmentofPreini1a1onComplexComponentsinYeast
12-44
RecruitmentofTFIID
• Acidictranscrip1on-ac1va1ngdomainoftheherpesvirustranscrip1onfactorVP16bindstoTFIIDunderaffinitychromatographycondi1ons
• TFIIDisrate-limi1ngfortranscrip1oninsomesystems
• TFIIDistheimportanttargetoftheVP16transcrip1on-ac1va1ngdomain
12-45
RecruitmentoftheHoloenzyme
• Ac1va1oninsomeyeastpromotersappearstofunc1onbyrecruitmentofholoenzyme
• Thisisanalterna1vetotherecruitmentofindividualcomponentsoftheholoenzymeoneata1me
• Someevidencesuggeststhatrecruitmentoftheholoenzymeasaunitisnotuncommon
12-46
RecruitmentModelofGAL11P-containingHoloenzyme
• Dimeriza1ondomainofGAL4bindstoGAL11Pintheholoenzyme
• A`erdimeriza1on,theholoenzyme,alongwithTFIID,bindstothepromoter,ac1va1ngthegene
Activation by Gal 11P and Gal 11-lex.
12-48
12.5Interac1onAmongAc1vators
• Generaltranscrip1onfactorsmustinteracttoformthepreini1a1oncomplex
• Ac1vatorsandgeneraltranscrip1onfactorsalsointeract
• Ac1vatorsusuallyinteractwithoneanotherinac1va1ngagene– Individualfactorsinteracttoformaproteindimerfacilita1ngbindingtoasingleDNAtargetsite
– SpecificfactorsboundtodifferentDNAtargetsitescancollaborateinac1va1ngagene
12-49
Dimeriza1on
• Dimeriza1onisagreatadvantagetoanac1vatorasitincreasestheaffinitybetweentheac1vatoranditsDNAtarget
• Someac1vatorsformhomodimersbutothersfunc1onasheterodimers
12-50
Ac1onataDistance
• Bacterialandeukaryo1cenhancerss1mulatetranscrip1oneventhoughlocatedsomedistancefromtheirpromoters
• Fourhypothesesaeempttoexplaintheabilityofenhancerstoactatadistance– Changeintopology– Sliding– Looping– Facilitatedtracking
12-51
HypothesesofEnhancerAc1on
12-52
3C:MethodtodetectDNAlooping
• Chromosomeconforma1oncapture(3C)isatechniqueusedtodetermineifenhancerac1onrequiresDNAlooping
• UsedtotestwhethertworemoteDNAregions,suchasanenhancerandapromoter,arebroughttogether
Chroma1nconforma1oncapture(3C)
12-54
GenomicImprin1ng
• Becausemosteukaryotesarediploidorganisms,youwouldpredictthatitdoesnotmaeerwhichalleleofanygivengenecameformthemotherorthefather
• Thisistrueinmostcasesbutthereareimportantexcep1ons
• Thedifferencesbetweenthegenesresidesinhowtheyaremodified,orimprinted,differentlyinfemalesandmales
• Evidenceexistsinmiceandhumans
Affinity chromatography transcription factor assay
Gal facilitates initiation complex formation.
Gal4s1mulatesPICforma1oninanuclearextract
TFIIB association with TFIID is stabilized by Gal4.
TFIIBassocia1onwithTFIIDisstabilizedbyGal4
TFIID and not TBP allows activator function in vitro.
Multiple sites for activator binding exhibit cooperativity in transcription and recruiting TFIIE.
Recruitment of holoenzyme through Gal 4-Gal11 interactions.
Leucine zippers.
DNA binding by the AP-1 family of activators use heterotypic dimers.
Metazoan Promoters are complex
Modular nature of enhancer elements in the sea urchin Endo 16 gene.
Integrative role of enhancer element A.
12-67
Enhanceosome
• Anenhanceosomeisanucleoproteincomplexcontainingacollec1onofac1vatorsboundtoanenhancerinsuchawaythats1mulatestranscrip1on
• ThearchetypalenhanceosomeinvolvestheIFNβenhancerwithastructurethatinvolveseightpolypep1desboundcoopera1velytoanessen1allystraight55-bpstrechofDNA
Human interferon gene promoter.
Insulator Function.
12-71
ModelofMul1pleInsulatorAc1on
Su(Hw)suppressorofhairywingisaninsulator
12-73
Summary
• Someinsulatorshavebothenhancer-blockingandbarrierac1vi1es,butsomehaveonlyoneortheother
• InsulatorsmaydotheirjobbyworkinginpairsthatbindproteinsthatcaninteracttoformDNAloopsthatwouldisolateenhancersandsilencerssotheycannolongers1mulateorrepresspromoters
• InsulatorsmayestablishboundariesbetweenDNAregionsinachromosome
12-74
SignalTransduc1onPathways
• Signaltransduc1onpathwaysbeginwithasignalingmoleculeinterac1ngwithareceptoronthecellsurface
• Thisinterac1onsendsthesignalintothecellandfrequentlyleadstoalteredgeneexpression
• Manysignaltransduc1onpathwaysrelyonproteinphosphoryla1ontopassthesignalfromoneproteintoanother
• Thisleadstosignalamplifica1onateachstep
Signaling cascade
12-76
Ubiquityla1on
• Ubiquityla1on,especiallymonoubiquityla1on,ofsomeac1vatorscanhaveanac1va1ngeffect
• Polyubiquityla1onmarksthesesameproteinsfordestruc1on
• Proteinsfromthe19Sregulatorypar1cleoftheproteasomecans1mulatetranscrip1on