分子生物学Chapter 11 Prokaryotic Gene Regulation.docx
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分子生物学Chapter 11 Prokaryotic Gene Regulation.docx
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分子生物学Chapter11ProkaryoticGeneRegulation
Chapter11ProkaryoticGeneRegulation
1.PrinciplesofOperonTheory
vAnOperonisasetofgenesthatareexpressedinunison,ietheyareCoordinatelyexpressed,togetherwiththeDNAcontrolelementsusedfortheirexpression.
vThegenesthatareCoordinatelyexpressedarecalledStructuralGenesandencodeproteinsthatfunctionenzymaticallyinacommonprocesssuchasabiosyntheticorcatabolicpathway.
theDNAcontrolelements
vThisOperatorbindingsitefortheRepressoristhemajortypeofDNAcontrolelement.
vAnegatively-actingproteincalledtheRepressorregulatesexpressionoftheOperonbybindingtoanOperatorDNAsitenearthepromoterfortranscription.
vThegenesthatencoderegulatoryproteinssuchasRepressorsarecalledRegulatorGenesandareNOTusuallypartofthesetofcoordinatelyexpressedoperongenes.
InducerandCo-Repressor
vAsmallmolecule(metabolite)usuallyinteractswiththeRepressor.ThissmallmoleculeiseitheranInduceroraCo-Repressor:
vAnInducerinactivatestheRepressorintheInducer-Repressorcomplex.
vACo-RepressoractivatestheRepressorintheInducer-Repressorcomplex.
Trans-actingFactorsandCis-actingelements
vProductsofRegulatoryGenescanthendiffuseormovetotheirtargetsandhenceareTrans-actingFactors.
vOperatorsandDNAcontrolelementsarethetargetsandaffectonlytheDNAtowhichtheyareattached;theyareCis-actingelements.AnyproteinbindingsiteonDNA,e.g.transcriptionpromotersandterminators,arecis-actingelements.
vExpressionofthegeneencodingtheRepressorisusuallyNOTitselfcontrolledbyaRepressor.Itthenisexpressedatalltimes,ieisconstitutivelyexpressed.
Chapter11ProkaryoticGeneRegulation
2.TwogeneralOperonClasses
2.1.CatabolicOperons
ThesmallmoleculeinteractingwiththeRepressorisanInducer,andisusuallythesugarormetabolicwhichisbrokendown.
Notetherationale:
theoperonshouldbeexpressedonlywhenInducerispresent,toexpressthegenesneededtobreakdown(catabolize)theInducer,egsugar
2.2.Anabolic(biosynthetic)Operons
vThesmallmoleculeinteractingwiththeRepressorisaCo-Repressor,andisusuallytheend-productofthebiosyntheticpathway,eganaminoacid.
vNotetherationale:
theoperonshouldbeexpressedonlywhenCo-Repressorisabsent,toexpressthegenesneededtosynthesizetheCo-Repressor,egaminoacid
3.LacOperon
Chapter11ProkaryoticGeneRegulation
3.1.BasicFeatures
vThreestructuralgenes:
lacZ-lacY–lacA
vencodingb-galactosidase,lacpermease,andlactransacetylaserespectively.
vb-galactosidasehydrolyzeslactosetogalactoseandglucose
permeasetransportslactoseintothecell;
andtransacetylaseisimportantonlyinsomeenvironments
vE.colicanuselactoseasasourceofcarbon.
vTheenzymesrequiredfortheuseoflactoseasacarbonsourceareonlysynthesizedwhenlactoseisavailableasthesolecarbonsource.
vGeneencodingLacRepressor:
lacI...activeasaTetramer
vfortuitouslyadjacenttoLacOperon...constitutivelyexpressed:
10copies/cell
vInducer:
b-galactosides,includingnon-metabolizablemoleculessuchasIPTG
Xgal:
acolorometricinducer...whencleavedyieldsabluecolor...
vInductionoccurswhenInducerisaddedtogrowthmedium:
Repressorisinactivated
v1000-foldincreaseingeneexpressioninminutes
withrapidreturntoBasalLevel(fewmoleculesexpressedunderrepression)whenInducerisremoved
Chapter11ProkaryoticGeneRegulation
3.2MutationsinRegulationProcesses
vInsightintothemechanismscontrollingsynthesisofβ-galactosidaseandlactosepermeasefirstcamefromthestudyofmutantsinwhichcontrolofβ-galactosidaseexpressionwasabnormal.Asensitivecolorimetricassayforβ-galactosidaseusesX-gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside)assubstrate:
3.2.1ConstitutiveMutants
vMutationsinwhichtheLacOperonisalwaysturnedon
v1)lacOcoperatorconstitutivemutants:
pointmutationsintheOperatorthatpreventRepressorbinding...Lacoperonisalwaysturnedon
vThesemutantsareCis-dominant,ietheyaffectONLYtheDNA(lacgenes)towhichtheOperatorisattached.
vThus,lacOc/lacO+merodiploidsarephenotypicallyLacOc,ieareconstitutive,butonlyviaexpressionofthelacgenesonthesameDNAasthelacOcmutation..
ExperimentaldemonstrationthatOcmutationsarecis-acting.(Cis:
wildphenotype)
ExperimentaldemonstrationthatOcmutationsarecis-acting.(Trans:
mutationphenotype)
v2)lacI-mutants
vDeletionsinlacI:
RepressorisinactiveandLacoperonison
ExperimentaldemonstrationthatthelacI+geneistrans-acting.(Cis:
wildphenotype)
ExperimentaldemonstrationthatthelacI+geneistrans-acting.(Trans:
wildphenotype)
3.2.2.NoninducibleMutants:
vMutationsthatpreventinduction,i.e.lacoperonalwaysturnedoff
v1)lacIsmutants:
pointmutationsthatyielda"SuperRepressor",onethatbindstheOperatoreveninthepresenceofInducer
v2)lacPmutants:
nearlyallPromotermutationsare"down"mutants,i.e.thatpromoterdoesnotsupporttranscriptionbyHoloRNApol.Inthiscase,thelacoperonisturnedoffindependentofpresenceorabsenceofInducer...
Chapter11ProkaryoticGeneRegulation
3.3MechanismofInhibitionofTranscription
vJacobandMonodmodeloftranscriptionalregulationofthelacoperonbylacrepressor
Chapter11ProkaryoticGeneRegulation
3.4BindingofLacRepressortoOperator
vOperatordefinedbylacOcmutants...26bpregion,nucpalindrome(inpart).
Equilibriumconstants
VirtuallyalltherepressorinthecellisboundtoDNA
Chapter11ProkaryoticGeneRegulation
3.5CataboliteRepression:
"glucoseeffect"
vWhenE.colifinds(forexample)bothglucoseandlactoseinthemedium,itmetabolizestheglucoseandrepresstheuseoflactose.Thischoiceisaccomplishedbypreventingexpressionofseveraloperons,includinglac,galandara.Thiseffectiscalledcataboliterepression.
Mechanism:
vWhenGlucoseispresent,cAMPlevelsarelow,andlowexpressionofLacoperonevenwhenLactoseispresent.
vWhenGlucoseisabsent,cAMPlevelsarehigh,andLactoseinducesLacoperontohighlevelofexpression
vAdenylatecyclaseconvertsATPintocAMP.
vPhosphodiesteraseconvertscAMPtoAMP.
vGlucosecatabolitemayinhibitadenylatecyclaseandstimulatePhosphodiesterase
Cataboliteactivatorprotein(CAP),butsometimesisreferredtoascAMP-receptorprotein(CRP).
vComplexofcAMP(3',5'-cyclic-AMP;)andCataboliteActivatorProtein(CAP)bindsasDimertoCAPbindingsiteusuallywithinoradjacenttoPromoterofCatabolicOperons,e.g.intheLacoperonpromoteratleftend.
vBindingofCAP-cAMPisrequiredforexpressionoftheseCataboliteOperons,i.e.TranscriptionoftheseOperonsisactivatedbyCAP-cAMPbinding
CAPBindingSite:
~22bp,pentamerpalindrome
vCAP-cAMPbindingaffectsbothRNApolymeraseandDNAstructure:
vCAP-cAMPinteractsdirectlywithAlphasubunitofRNApolymerase...geometryofthepreciselocationoftheCAPbindingsiteisimportanthere(operonsdiffer...)
v
vCAP-cAMPbindingbendstheDNAnearly90°
vNegativeandpositivetranscriptionalcontrolofthelacoperonbythelacrepressorandcAMP-CAP,respectively.
4.TheLuxoperon
Aspeciesofsquid
vAlightorgan,inwhichitculturesaverypure,verydensepopulationofabacteriacalledVibriofischeri.
vThisbacteriaproducesasubstancecalledluciferase,whichglowswiththesameintensityasthemoon,renderingthesquidinvisibletopredatorsfromthedepthsoftheocean.
vWhenVibriofischeriisnotinthesquid'slightorgan,itdoesnotneedtobemakingluciferase,sinceglowingwillnothelpitoranythingelse.Ontheotherhand,wheninsidethelightorgan,itistothebacteria'sadvantagetoglow,becausethenthesquidwillnotgeteatenandwillfeedit,awayfromcompetitionfromanyotherkindsofbacteria.
luxRisatranscriptionalactivatoroftheluxoperon.
vThegenesformakingluciferasearecontainedintheluxoperon.ADNAbindingsite(luxO)neartheluxpromoter(luxP)bindsaproteincalledluxR.ThisproteinsomehowcallsRNApolymeraseoverwhenitisboundtotheDNA,thusincreasingtranscriptionoftheDNA
Theconceptofquorumsensing
vEachbacteriumiscontinuouslysecretingauniquesmallmoleculecalledVAI(Vibriofischeriautoinducer)thatcandiffusereadilythroughthecellmembrane.Thus,thereisadecliningconcentrationofthesmallmoleculeinagrowingcircumferencearoundthebacterium.Whentherearemanybacteriaaround,thelocalconcentrationofthesmallmoleculewillbeveryhigh.
5.ArabinoseOperon
v1).CoordinatedexpressionofArabinoseIsomerase,ribulokinase,andanepimeraseinresponsetoarabinose"diet"inE.Coliinabsenceofglucose.(AraA,AraB,AraD).
v
ThearabinoseandCproteinoperonofE.coli
Regulatorysitesofthearabinoseoperon
vTwooperators(AraO1andAraO2)controlexpressionoftheBADgenes.
vArabinoseisrequiredforgoodexpression,glucosewilleliminateexpressioneveninthepresenceofarabinose.
vAcisregionresponsivetoarabinosefeedingwasidentified(AraI)andmapped
vAtranselementresponsivetoarabinosefeedingwasidentified(AraC)andmapped.Thiselementcodesforthearabinosebindingprotein(repressor)
vACAPbindingsiteforthecAMPregulatedcataboliterepressor(asfoundfortheLacOperonwasalsofound(CAPSite).
AraCcodesforaprotein(C-protein)whichmediatesrepressionofBADexpressio
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