chapter 9 -transcriptional control of gene expression · pdf file07.04.2017 ·...
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Chapter9- TranscriptionalControlofGeneExpression
Chapter9- TranscriptionalControlofGeneExpression
9.1ControlofGeneExpressioninBacteria9.2OverviewofEukaryoticGeneControl9.3RNAPolymeraseIIPromotersandGeneralTranscriptionFactors9.4RegulatorySequencesinProtein-CodingGenesandtheProteinsThroughWhichTheyFunction9.5MolecularMechanismsofTranscriptionRepressionandActivation9.6RegulationofTranscription-FactorActivity9.7EpigeneticRegulationofTranscription9.8OtherEukaryoticTranscriptionSystems
InteractwithoutsideworldBehavior– Output– Change
Environmentalchange– Behavioralchange…
CelllevelMoleculelevel
Organlevel
Behaviorlevel
Interactwithoutsideworld
CelllevelMoleculelevel
What’sthebasicproductofcell?
ProteinsGene
Genome
Howtoregulatetheproteinexpression?
Environmentalchanges
1. Transcription
1. Translation
1. Proteindegradation
1. RNAdegradation
Interactwithoutsideworld
CelllevelMoleculelevel
What’sthebasicproductofcell?
ProteinsGene
Genome
What’sthemostefficientwaytoregulatetheproteinexpression?
Environmentalchanges
Interactwithoutsideworld
CelllevelMoleculelevel
What’sthebasicproductofcell?
ProteinsGene
Genome
What’sthemostefficientwaytoregulatetheproteinexpression?
EnvironmentalchangesTranscriptionalregulation!Howpercentageisit?
Contributionsofthemajorprocessesthatregulateproteinconcentrations.
Whenisthetranscriptionalregulationimportant?
• Interactwithoutsideworld• ?• Formakingyourownshape…
Developmentalprocess…
DNAmicroarray
Geneexpressionchangesinmicroarraystudy
Phenotypesofmutationsingenesencodingtranscriptionfactors.
• Improperregulationofmechanismscontrollingtranscriptioncausespathologicalprocesses.
• (a)AhumanHOXD13 genedominantmutation:• polydactyly– developmentofextradigits
• (b)DrosophilaUbx genehomozygousrecessivemutations:
• preventUbx expressioninthethirdthoracicsegment
• transformssegment,whichnormallyhasabalancingorgancalledahaltere,intoasecondcopyofthethoracicsegmentthatdevelopswings
• (c)InactivatingmutationsinbothcopiesofthreeArabidopsisthalianafloralorgan–identitygenes:
• transformthenormalflowerpartsintoleaflike structures
• Mutationsaffectmasterregulatorytranscriptionfactorsthatregulatemultiplegenes,includingmanygenesencodingothertranscriptionfactors.
Howtoregulatethetranscription?
TranscriptionalregulationinBacteria
Learningtheprinciples…
TranscriptionalControlofGeneExpression
9.1ControlofGeneExpressioninBacteria• Prokaryotegeneexpression– regulatedprimarilybymechanismsthatcontrolgenetranscription
• Thelacoperonandsomeotherbacterialgenesareregulatedbyactivatorproteinsthatbindnexttoapromoter andactivateRNApolymerase.
• TranscriptioninitiationbyE.coliRNApolymerasescanberegulatedbyrepressorsandactivatorsthatbindnearorupto100basesupstreamofthetranscriptionstartsite.
• Transcriptioninbacteriacanberegulatedbycontroloftranscriptionalelongationinthepromoter-proximalregion.
Tounderstandthetranscriptionalregulationofanorganism,
• Weshouldknowtheorganism.
• Weshouldknowtheenvironmentaroundtheorganism.
Interaction!
E.coli
• Escherichiacoli• Escherichiacoli isa gram-negative, facultativelyanaerobic, rod-shaped, coliformbacterium ofthe genus Escherichia thatiscommonlyfoundinthelower intestine of warm-blooded organisms(endotherms)
Symbiosis
Gutflora (gutmicrobiota,or gastrointestinalmicrobiota)isthecomplexcommunityof microorganisms thatliveinthe digestivetracts ofhumansandotheranimals,includinginsects.
Intestinal bacteria alsoplayaroleinsynthesizing vitaminB and vitaminK aswellasmetabolizing bileacids, sterols,and xenobiotics.
JustlearnabouttheE.coli
• Gram-negative
• Facultatively anaerobic
Gram-negativebacteria areagroupof bacteria thatdonotretainthe crystalviolet stain usedinthe Gramstaining methodofbacterialdifferentiation.
JustlearnabouttheE.coli
• Gram-negative
• Facultatively anaerobic
Gram-negativebacteria areagroupof bacteria thatdonotretainthe crystalviolet stain usedinthe Gramstaining methodofbacterialdifferentiation.
JustlearnabouttheE.coli
• Gram-negative
• Facultatively anaerobic
Gram-negativebacteria areagroupof bacteria thatdonotretainthe crystalviolet stain usedinthe Gramstaining methodofbacterialdifferentiation.
A facultativeanaerobe isan organism thatmakes ATP by aerobicrespiration if oxygen ispresent,butiscapableofswitchingto fermentation or anaerobicrespiration ifoxygenisabsent.
RegulationoftranscriptionfromthelacoperonofE.coli.
• E.coliinanenvironmentthatlackslactose:
• E.coliinanenvironmentcontainingbothlactoseandglucose:
• E.coliinanenvironmentcontaininglactosebutlackingglucose:
Lactosemetabolism
RegulationoftranscriptionfromthelacoperonofE.coli.
• E.coliinanenvironmentthatlackslactose:represslacmRNAsynthesissothatcellularenergyisnotwastedsynthesizingunnecessaryenzymes.
• E.coliinanenvironmentcontainingbothlactoseandglucose:cellspreferentiallymetabolizeglucose,thecentralmoleculeofcarbohydratemetabolism.
• E.coliinanenvironmentcontaininglactosebutlackingglucose:cellsmetabolizelactoseatahighrateonlywhenlactoseispresentandglucoseislargelydepletedfromthemedium.
Transcriptionalregulation
• Thinkabouttranscription
HowtoblockthebindingofRNApolymerase?
Howtocontrolthetranscription?1.Blockthetranscription
Howtocontrolthetranscription?1. Blockthetranscription2. Activatethetranscription
Transcriptionalregulation
• Cis-actingelement• Trans-actingelement
Promoter
Protein
RegulationoftranscriptionfromthelacoperonofE.coli.
• CAPsite– bindscataboliteactivatorprotein.• lacpromoter– bindstheσ70-RNApolymerasecomplex
• lacoperator– bindslacrepressor
cAMP-bound cataboliteactivatorprotein(CAP)
RegulationoftranscriptionfromthelacoperonofE.coli.
• Tetramericlacrepressor:bindstotheprimarylacoperator(O1)andoneoftwosecondaryoperators(O2orO3)simultaneously.Thetwostructuresareinequilibrium.
Lacoperon
https://www.youtube.com/watch?v=oBwtxdI1zvk
• MostE.colipromotersinteractwithσ70-RNApolymerase,themajorformofthebacterialenzyme.
DNAloopingpermitsinteractionofboundNtrC andσ54-RNApolymerase.
regulatedsolelybyactivatorsthatbindtoenhancersitesgenerallylocated80–160bpupstreamfromthetranscriptionstartsite
• NtrC σ54-activator:• NtrC protein(nitrogenregulatory
proteinC)– stimulatestranscriptionoftheglnA gene
• glnA gene– encodesglutaminesynthetase,whichsynthesizesglutamine,thecentralmoleculeofnitrogenmetabolism,fromglutamicacidandammonia
• NtrC ATPhydrolysis– requiredforactivationofσ54-RNApolymerase
Enhancer
The σ54-RNA polymerase binds to the glnA promoter but does not melt the DNA strands and initiate transcription until it is activated by NtrC, a dimeric protein.
Howtodetectamountofasubstanceandregulatetheamount?
?
Regulator SubA synthesisenzyme
Howtodetectasubstanceandregulate?
BindingtoSubA SubA synthesisenzyme
Howtodetectasubstanceandregulate?
BindingtoSubA SubA synthesisenzyme
Howtodetectasubstanceandregulate?
BindingtoSubA SubA productiongeneactivator
Production
Activate
Howtodetectasubstanceandregulate?
BindingtoSubA
Production
Activate
Two-componentregulatorysystems.
• Manybacterialresponsesarecontrolledbytwo-componentregulatorysystems.
• (a)Lowcytoplasmicglutamineconcentration:• glutaminedissociatesfromNtrB.• NtrB conformationalchangeactivatesaproteinkinasetransmitterdomainthattransfersanATPγ-phosphatetoaconservedhistidine(H)inthedomain.
• Phosphate– thentransferredtoanasparticacid(D)intheregulatorydomain oftheresponseregulatorNtrC
• PhosphorylationconvertsNtrC intoitsactivatedform,whichbindstheenhancersitesupstreamoftheglnApromoter.
Generaltwo-componentsignalingsystem
• (b)Two-componenthistidyl-aspartylphospho-relayregulatorysystemsinbacteriaandplants– generalorganization
Otherwaytoregulatethetranscription
Transcriptionalregulation
• Transcriptioninitiation,then?
Elongation&Termination
Trp operon
But,stillleakofexpression
Calledas“attenuation”
Howtoblocktheexpressioncompletely?
TranscriptionalcontrolbyregulationofRNApolymeraseelongationandterminationintheE.colitrp operon.
• Ribosometranslatesquicklythroughregion1intoregion2,blockingregion2basepairingwithregion3.
• Region3basepairswithregion4.• 3–4stem-loopandfollowingseriesofuracils – signal
forterminationofRNApolymerasetranscriptionofrestofoperon
• Region3issequesteredinthe2–3stem-loopandcannotbase-pairwithregion4.
• Transcriptionofthetrp operoncontinues.
1.Nocodingforenzymes(~140nts)
2.Trp-Trp codons
3.
4.Indicatetermination
Riboswitchcontroloftranscriptiontermination inB.subtilis. Bacillussubtilis,knownalsoasthe haybacillus or grassbacillus,is
a Gram-positive, catalase-positive bacterium
xpt-pbuX operon(encodesenzymesinvolvedinpurinesynthesis)
TranscriptionalControlofGeneExpression
9.2OverviewofEukaryoticGeneControl• Transcriptionalcontrolistheprimarymeansofregulatinggeneexpression
ineukaryotes.• Eukaryotescontainthreetypes ofnuclearRNApolymerases:
– RNApolymeraseIsynthesizesonlypre-rRNA.– RNApolymeraseIIsynthesizesmRNAs,someofthesmallnuclear
RNAsthatparticipateinmRNAsplicing,andmicro- andsmallinterferingRNAs(miRNAs andsiRNAs)thatregulatethetranslationandstabilityofmRNAs.
– RNApolymeraseIIIsynthesizestRNAs,5SrRNA,andseveralothersmallstableRNAs.
Fundamentaldifferencebetweeneukaryoticandprokaryoticchromatin?
• SinglehumancellDNAmeasuresabout2mintotallength,andiscontainedwithinnucleiwithdiametersoflessthan10μm – acompactionratioofgreaterthan105 to1.
• EachchromosomeconsistsofasingleDNAmolecule(aslongas280Mbinhumans),organizedintoincreasinglevelsofcondensationfromnucleosomestohigherorderchromatinfoldingbyhistoneandnonhistone proteins.
• AnygivenportionofhighlycompactedDNAcanbeaccessedfortranscription,replication,andrepairofdamagewithoutthelongDNAmoleculesbecomingtangledorbroken.
• HighereukaryoticDNAconsistsofuniqueandrepeatedsequences:
• Onlyabout1.5percentofhumanDNAencodesproteinsandfunctionalRNAs.
• Theremainderincludesregulatorysequencesthatcontrolgeneexpressionandintrons.
• About45percentofhumanDNAisderivedfrommobileDNAelements,geneticsymbiontsthathavecontributedtotheevolutionofcontemporarygenomes.
Overviewofeukaryotictranscriptionalcontrol.
• Chromatincondensationinactivatesgenetranscription:• BlocksRNApolymerasesandgeneraltranscriptionfactors
frominteractingwithgenepromoters• Repressorproteins:
• Maybindtotranscription-controlelementstoinhibittranscriptioninitiationbyPolII
• Mayinteractwithmultiproteinco-repressorcomplexestocondensechromatin
• Pioneertranscriptionfactor:• Bindstoaspecificregulatorysequencewithinthe
condensedchromatin• Interactswithchromatin-remodelingenzymesandhistone
acetylases thatdecondense thechromatin,makingitaccessibletoRNApolymeraseIIandgeneraltranscriptionfactors.
• Activatorproteins:• Bindtospecifictranscription-controlelementsinboth
promoter-proximalsitesanddistantenhancers• Interactwithoneanotherandwiththemultisubunit
MediatorcomplextoassemblegeneraltranscriptionfactorsandRNApolymeraseII(PolII)onpromoters
• Transcriptionalactivation:• PolIIinitiatestranscription.• PolIIpausesaftertranscribingfewerthan100nucleotides
becauseofactionoftheelongationinhibitorNELF(negativeelongationfactor)associatedwithDSIF(DRBsensitivity-inducingfactor).
• Activators:• PromoteassociationofthePolII-NELF-DSIFcomplexwith
elongationfactorP-TEFb (cyclinT-CDK9[kinase]),whichreleasesNELF
• NELFreleaseallowsresumptionofRNAtranscription.
Pax 6gene
Pax6protein– requiredfordevelopmentoftheeye,certainregionsofthebrainandspinalcord,andthepancreascellsthatsecretehormonessuchasinsulin
regulation at each step m gene expression have been found, control of transcript ion initiation and elongation-the first rwo steps-arc the most important mechanisms for determining whether most genes are expressed and how much of the en-coded mRNAs and, consequently, proteins are produced. The molecular mechanisms that regulate transcription initiation and elongation are critical to numerous biological phenomena, in-cluding the development of a multicellular organism from a single fertilized egg cell as mentioned above, the immune re-sponses that protect us from pathogenic microorganisms, and
(a)
(b)
Haltere
Normal
(c)
FIGURE 7·1 Phenotypes of mutations in genes encoding transcription factors. (a) A mutation that inactivates one copy of the Pax6 gene on either the maternal or paternal chromosome 9 results in failure to develop an iris, or aniridia. (b) Homozygous mutations that prevent expression of the Ubx gene in the third thoracic segment of Drosophila result in transformation of t he third segment, which normally has a balancing organ called a haltere, into a second copy of the thoracic segment that develops wings. (c) Mutations in Arabidopsis
280 CHAPTER 7 • Tran scriptional Control of Gene Expression
neurological processes such as learning and memory. When these regulatory mechanisms contro ll ing transcription function improperly, pathological processes may occur. For example, reduced activity of the Pax6 gene causes aniridia, failure to de-velop an iris (Figure 7-la) . Pax6 is a transcription factor that normally regulates transcription of genes involved in eye devel-opment. In other organisms, mutations in transcription factors cause an extra pair of wings ro develop in Drosophila (Figure 7-1 b), alter the structures of flowers in plants (Figure 7- l c), and are responsible for multiple other developmental abnormalities.
Ubxmutant
thaliana that inactivate both copies of t hree flora l organ-identity transform the normal parts of the f lower into leafl ike structures. In each case, these mutations affect master regulatory transcription factors that regulate multiple genes, including many genes encoding other transcription factors. [Part (a), left, Simon Fraser/Photo Researchers, Inc.; right, Visuals Unlimited. Part (b) from E. B. Lewis, 197B, Nature 276:565. Part (c) from D. Wiegel and E. M. Meyerowiu, 1994, Ce//78:203.]
A mutation that inactivates one copy of the Pax6 gene on either the maternal or paternal chromosome 9 results in failure to develop an urus, or aniridia.
Transcription-controlregionsofthemousePax6geneandtheorthologoushumanPAX6gene
• ThreealternativePax6promoters:• Transcription-controlregions(200–500bp long)regulateexpressionofPax6 atdifferentembryogenesistimesinindifferenttissues.
Betagalactosidase– asareporterfortranscriptionalregulation
Promoter Reporter
B-galactosidase
Experiment:tg mouseexpressingofabeta-galactosidasereportertransgenefusedtodifferentregionsofPas6gene
• Experiment:transgenicmouseexpressionofaβ-galactosidasereportertransgenefusedtodifferentregionsofPax6 gene(stainedwithX-galtorevealβ-galactosidase)
• (b)Embryo10.5daysafterfertilization– 8kbofmouseDNAupstreamfromexon0linkedtoreportertransgene
• Expression– inlenspit(LP)tissuethatwilldevelopintotheeyelensandintissuethatwilldevelopintothepancreas(P)
• (c)Embryo13.5daysafterfertilization– sequencebetweenexons4and5linkedtoβ-galactosidasereportergene
• Expression– Retinaandpancreas
(b)
(c)
ThehumanSALL1enhanceractivatesexpressionofareportergeneinlimbbudsofthedevelopingmouseembryo
HumanDNAsequence~500kbdownstreamoftheSALL1gene– highlyconservedbetweenhumans,mice,chickens,frog,andfish.
ThehumanSALL1enhanceractivatesexpressionofareportergeneinlimbbudsofthedevelopingmouseembryo
Beta-galpromoter
Plasmidvector
Luciferase
• Luciferase isagenerictermfortheclassofoxidativeenzymesthatproducebioluminescence,andisdistinctfromaphotoprotein.
ThreeRNApolymerases
HowtoseparateRNApolymerases
LiquidchromatographyseparatesandidentifiesthethreeeukaryoticRNApolymerases,eachwithitsownsensitivityto𝛂-amanitin.
AllthreeRNApolymerasesinaproteinextractfromculturedeukaryoticcellnucleiboundtoanegativelychargedDEAESephadex column
RNApolinhibitor
α-Amanitin (red)boundtoRNApolymeraseIIfrom Saccharomycescerevisiae (brewer'syeast).
α-Amanitin isanselectiveinhibitorof RNApolymeraseII and III. Thismechanismmakesitadeadlytoxin.
interfereswiththetranslocationofRNAandDNAneededtoemptythesiteforthenextroundofRNAsynthesis
LiquidchromatographyseparatesandidentifiesthethreeeukaryoticRNApolymerases,eachwithitsownsensitivityto𝛂-amanitin.
• RNApolymeraseI:• insensitive• locatedinthenucleolus• transcribesgenesencodingprecursorrRNA (pre-rRNA),whichisprocessedinto28S,5.8S,and18SrRNAs
• RNApolymeraseII:• verysensitive• transcribesallprotein-codinggenestoproducemRNAs
• RNApolymeraseIII:• intermediatesensitivity• transcribesgenesencodingtRNAs,5SrRNA,andanarrayofsmallstableRNAs,includingoneinvolvedinRNAsplicing(U6)andtheRNAcomponentofthesignalrecognitionparticle(SRP)
AllthreeRNApolymerasesinaproteinextractfromculturedeukaryoticcellnucleiboundtoanegativelychargedDEAESephadex column
Comparisonofthree-dimensionalstructuresofbacterialandeukaryoticRNApolymerases
• Fourmultisubunit RNApolymeraseshaveasimilaroveralldesign.• EachofthethreeeukaryoticRNApolymerasesismorecomplexthanE.coliRNApolymerase
• (a)BacterialRNApolymerase• (b)EukaryoticRNApolymeraseII(10ofthe12subunitsshowninmodel)• (c)YeastRNApolymeraseII– includingsubunits4and7thatextendfromthecoreportionoftheenzymeshownin(b)neartheregionoftheC-terminaldomainofthelargesubunit
Evolution?
RNApolIIsubunitsandstructure
https://www.youtube.com/watch?v=GdKfadJGId4
TheclampdomainofRPBI(RNApolB1=RNApolII).• DiffersfromfreemainlyinthepositionofaclampdomainintheRPB1subunit
• Clampdomainswingsoverthecleftbetweenthejawsofthepolymeraseduringformationofthetranscribingcomplex,trappingthetemplateDNAstrandandtranscript.
• Bindingoftheclampdomaintothe8–9-bpRNA-DNAhybrid– mayhelpcoupleclampclosuretothepresenceofRNA
• Mg2+ion– participatesincatalysisofphosphodiesterbondformation
• RPB2Walldomain– forcesthetemplateDNAenteringthejawsofthepolymerasetobendbeforeitexitsthepolymerase
• Bridgeαhelix:• extendsacrossthecleftinthepolymerase
• ispostulatedtobendandstraightenasthepolymerasetranslocates onebasedownthetemplatestrand
• Nontemplate strand– formsaflexiblesingle-strandedregionabovethecleft(notshown),extendingfromthreebasesdownstreamofthetemplatebase-pairedtothe3ʹbaseofthegrowingRNAtowherethetemplatestrandexitsthepolymerase
SchematicrepresentationofthesubunitstructureoftheE.coliRNAcorepolymeraseandyeastnuclearRNApolymerases.
Allthreeyeastpolymeraseshavefivecoresubunitshomologoustotheβ,βʹ,twoα,andω subunitsofE.coliRNApolymerase
RNApolymerasesIandIIIcontainthesametwononidentical α-likesubunits,whereasRNApolymeraseIIcontainstwoothernonidentical α-likesubunits.
• RNApolymeraseIIlargestsubunit(RPB1)– alsocontainsanessentialC-terminaldomain(CTD)
CTDofRNApolII
• RNAPolymeraseIIexistsintwoformsunphosphorylated andphosphorylated,IIAandIIOrespectively.
• ThemethodfortheelongationinitiationisdonebythephosphorylationofSerineatposition5(ser5),viaTFIIH.ThenewlyphosphorylatedSer5recruitsenzymestocapthe5'endofthenewlysynthesizedRNAandthe"3'processingfactorsto poly(A) sites".
CTDofRNApolIIinaction!Cracking the RNA polymerase II CTD codeSylvain Egloff, Shona Murphy <Find the paper!>
Antibodystainingdemonstratesthatthecarboxy-terminaldomainofRNApolymeraseIIisphosphorylated duringinvivotranscription.
• PhosphorylatedCTD(red)–stainedwithaphosphorylation-specificrabbitantibody
• Nonphosphorylated CTD(green)– stainedwithnonphosphorylation-specificgoatantibody
Whatcanyousee?
Polytene chromosomes areoversized chromosomes whichhavedevelopedfromstandardchromosomesandarecommonlyfoundinthesalivaryglandsof Drosophilamelanogaster.
Discussionwithfriends
• PleasefindthefunctionsofRNAs
• SummarizetherolesofCTDinRNAPolII-mediatedtranscription