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