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Lecture12 Gene,cs BIOL335 Pleasesignana*endancesheet Classstartsat4pmsharp! Assignmentsreturnedinlabsec8on Con8nuemappinggenesinbacteria Generegula8oninbacteria -JacobandMonodandthelacoperon Quizgrading Bacterialsex(conjuga(on) StrainA: met bio-thr+leu+thi+ StrainB: met+bio+thr-leu-thi- mixture Discoveredby 21yearoldJoshua Lederberg(1946) Washandplate Washandplate Washandplate 108cells 108cells 108cells minimalmedia No colonies minimalmedia minimalmedia No Colonies!!! colonies DNAistransferred betweenbacteriaviapili TransferofF+plasmidfromdonortorecipient bacterial chromosome Fplasmid F+donorcell F-recipientcell TransferofF+plasmidfromdonortorecipient F+cellextendspilustoF-cell F-plasmidisnicked,andasingle strandispassedthroughthepilus totherecipientcell TransferofF+plasmidfromdonortorecipient strandsarereplicated inbothcell F-plasmidtransfercomplete F-cellisnowF+!!! Conjuga8onbyHfrdonorcellstorecipients (F-factorconjuga8ongenesintegratedintodonorgenome) F-containingchromosomeis nickedattheFlocus,andasingle strandispassedthroughthepilus totherecipientcell DNAistransferredun8lcomplete, orun8lthepilusfallsapart Need~2hrsfortheen8re chromosometogettransferred ElieWollmanandFrancoisJacob(1957): useHfrcrossestomakemapsofbacteria chromosomes HfraziRgal+tonRstrS x Resistanttoaziandtondrugs, cangrowongalactose, killed(Sensi8ve)bystreptomycin F-aziSgal-tonSstrR killedbyaziandtondrugs, cannotgrowongalactose, resistanttostreptomycin letcrossfor2hrs getcellsthatareresistanttoallthreedrugs,can growongalactose,andcanactasHfrdonors ElieWollmanandFrancoisJacob(1957)–use Hfrcrossestomakemapsofbacteria chromosomes Puangamixtureofconjuga8ngbacteriathrougha blenderbreaksthepili,stoppingDNAtransfer Calledthe"coitusinterruptus"assayacerthe Va8can-approvedbirthcontrolmethod ElieWollmanandFrancoisJacob–useHfr crossestomakemapsofbacteriachromosomes HfraziRgal+tonRstrS x F-aziSgal-tonSstrR Systema8callyinterruptconjuga8onatvarious8mes andassayforrecombinants. PlateonstreptomycintokillHfrdonorcells ElieWollmanandFrancoisJacob–useHfr crossestomakemapsofbacteriachromosomes HfraziRgal+tonRstrS x F-aziSgal-tonSstrR ElieWollmanandFrancoisJacob–useHfr crossestomakemapsofbacteriachromosomes HfraziRgal+tonRstrS azi 5min 10min x F-aziSgal-tonSstrR gal ton F 12min 25min 2hrs Blendt=5min–norecombinantsseen Blendt=10min–Findaziresistantcells,butnotothers Blendt=12min–Findaziandtonresistantcells Blendt=25min–azi,tonresistanceandgalactose growthseen Blendt=2hrs–Seeazi,tonresistant,galactose growingcellsthatcanbeFdonors(completetransfer) WollmanandJacobusedmethodtomap severalEcolistrains O=origin,F=fer8lityfactor Eachstrainhadadifferentorderofgenes!?!? Intwostrains,thehisgeneistransferredbeforetheglygene(hisis closertoO),butinthreestrains,theglygeneistransferredbeforethe hisgene. AllanCampbellhypothesizedthatbacteriahave circularchromosomes,andthatthedifferentHfr strainshaveF integratedatdis8nct loca8ons AtransferredlinearHfrfragment(exogenote)must recombineintotherecipientchromosometoremain X exogenote double- recombina,on endogenote linearexogenote islost recombined into chromosome Moredetailedmappingofbacterialchromosomeswith recombina8onfrequencies,aswitheukaryotes Caveat:recombina8onfreqscanbemeasuredonlyfor genesonaexogenotefragmentthathasbeentransferredover Moredetailedmappingofbacterialchromosomeswith recombina8onfrequencies,aswitheukaryotes exogenote leu+ arg- met- chromosome leu+ arg+ met+ leu+ arg- met+ Gene8cmapofEcoli coitusinterruptus andrecombina8on mapsassembled intoagene8cmap InsteadofcM,units are"minutes"– conjuga8ontransfer8me Some8mesanintegratedF-factorplasmidcan popoutofthechromosome F-plasmid excision Hfrcell F+cell Some8mestheF-factorcanpopoutofthe chromosomeandtakesomeDNAalong–messy orimpreciseexcision Messyexcision; Result:F'plasmidthat hasadonatablelacgene, alacepisome Diploidjustforthelacgene F'episomesareveryveryveryusefulfor bacterialgene8cs -Canbeusedtotransferallelesfromonestrainto another -Canbeusedtoseewhetheragivenalleleis dominantorrecessive–canmakepar8aldiploids (merodiploids) FrancoisJacob&JacquesMonod(Nobel1965) usedF'episomestodiscoverhowbacterial genesareregulated "WhatistrueforE.coliistruefortheelephant” -JacquesMonod FrancoisJacob -StartedmedicalschoolbeforeWW2 -FledtoEnglandin1940 -JoinedtheFreeFrenchArmyunderDeGaulle -Injuredwhilefigh8ngNazis,awardedCroixdeGuerre -Finishedmedschool,butwaspassionateaboutbasicresearch. -JoinedthePasteurIns8tutewherehemet... -Americanmother,Frenchfather WorkedwithTHMorganintheUSbeforegradschool -StartedgradschoolinParis...thentheNazisinvaded -One8me,theGestapostoppedbyhislab...theyseemed uncomfortablewithallthebacterialcultures -Hisnightjob:ChiefofStaffoftheFrenchResistance HelpedassassinateNaziofficialsinOccupiedFrance,smuggled weapons,bombedrailroadstoslowGermantroops,etc -AcerWW2,befriendedfellowan8-Sovietlecistex-resistance member,theexisten8alistphilosopherAlbertCamus (TheStranger,MythofSisyphus,LiteratureNobel1957) Jacques Monod ChanceandNecessity(1970) Aninfluen8ala*empttoconnect molecularbiology,evolu8on, philosophy,andreligion... alessobnoxious,moreFrench predecessorofRichardDawkins "Randomnesscaughtonthewing,preserved, reproducedbythemachineryofinvariance andthusconvertedintoorder,rule,and necessity.Atotallyblindprocesscanby defini8onleadtoanything;itcanevenleadto visionitself.” “Manatlastknowsthatheisaloneintheunfeelingimmensityof theuniverse,outofwhichheemergedonlybychance.Hisdes8nyis nowherespelledout,norishisduty.Thekingdomaboveorthe darknessbelow;itisforhimtochoose." Ecoliproducestheenzymeβ-galactosidase (β-gal)tometabolizelactosesugar Ecolialsomakesatransporterprotein,lacpermease, totransportthesugarintothecytoplasm Ecoliproducestheenzymeβ-galactosidase (β-gal)tometabolizelactosesugarONLY whenlactoseisaround -Savesmaterialandenergy....wastefultomakean enzymeifitssubstrateisnotaround -IfglucoseANDlactoseareavailable,Ecolimakes theenzymeonlyaceralltheglucose,itspreferred food,isusedup -Goodmodelsystemtostudygeneregula8on: bothposi8veandnega8vecontrol β-galactosidaseiseasilyassayedwithsynthe8c substratesthatchangecoloruponcleavage H β-galactosidase Ortho-Nitrophenyl-β-galactoside (ONPG) colorless yellow + H Non-"edible"lactoseanalogscalledinducerscan trickEcolitoincreaseβ-gallevels1000-fold Inducer removed Inducer added isopropyl-b-D-thiogalactoside (IPTG) Usingthebacterialgene8csbeingdevelopedin the1950's,JacobandMonodiden8fiedand mappedanumberofgenesandmutantswith abnormalβ-galregula8on structuralgenes lacI lacZ lacY lacA permease transacetylase "theinducer" β-gal lacI-cellsproducethe enzymesallthe,me (cons(tu(vely) theseenzymesare co-regulated,solacZ ac8vityisagoodproxy lacI-wastheveryfirstmutanteverfoundtohavea defectnotintheac8vityofanenzyme,butinthe controlofenzymeproduc8on: thismuta8onINCREASEDac8vity! structuralgenes lacI lacO lacZ lacY lacA "theoperator" Theyalsodiscoveredasetofmuta8onsinagene,lacO thatmappedveryclosetolacZ Muta8onsonlacOwerealsocons8tu8veorfailed toproduceanyenzyme,dependingontheallele Fine-mappingoflacOmuta8onsrevealthey comeindis8nctclustersforeachclass structuralgenes lacO lacOnon-func8onal muta8onsmaphere lacZ lacY lacA lacOcons8tu8ve(Oc) muta8onsmaphere "promoter" lacP lacO lacZ "operator" lacY lacA FrancoisJacob&JacquesMonodused F'episomescontaininglacgenesandmutants Messyexcision; Result:F'plasmidthat hasadonatablelacgene, alacepisome Diploidjustforthelacgene lacOactsincis;controlonlygenesonsame lacZac8vity lacYac8vity chromosome Non- Induced induced O+ Z+ Y+ − + Conclusion:wild-typesystemisinducible O+ Z+ Y+/Fʹ O+ Z− Y+ − Conclusion:Z+isdominanttoZ− Oc Z+ Y+ + Conclusion:Ociscons8tu8ve Non- induced Induced − + + − + + + + + − O+ Z− Y+/Fʹ Oc Z+ Y− + Conclusion:OperatorlacOiscisac8ng; controlsonlyallelesonthesamechromosome + Calledcisandtransbyanalogy toOrganicChemistry cis-2-butene methylgroupson sameside ofthebond trans-2-butene methylgroupson oppositesides ofthebond lacIactsintrans;controlslac lacZac8vity genesanywhere lacYac8vity Non- Non- Induced induced induced I+ Z+ Y+ − + − Conclusion:wild-typesystemisinducible I− Z+ Y+ + + + Conclusion:I−iscons8tu8ve;lacIisrequiredforinducibility I+ Z− Y+/Fʹ I− Z+ Y+ − Conclusion:I+isdominanttoI− Induced + + + − + + − + I− Z− Y+/Fʹ I+ Z+ Y− − Conclusion:I+istransac8ng;itcanconferinducibilitytoagene regardlessofitschromosomalloca(on(sameordifferentchromosome) Someraremuta8onsoflacI(Is)neverproduce anylactoseenzyme "promoter" lacI "theinducer" structuralgenes lacP lacO lacZ lacY lacA "operator" beta-gal permease I-=cons8tu8veallele trans- acetylase Is="silent"allele;enzymesnevermade -Whenmapped,noclusteringofI-andIswithin thelacIgene lacIsmutantallelesalsoactintrans;cellsthat haveacopyneverproduceanylacenzymes lacZac8vity lacYac8vity Non- Non- Induced Induced induced induced Is Z+ Y+ − − − − Conclusion:lacIsresultsincons8tu8verepression(neverturnson) IsZ+ Y+/Fʹ I+Z+Y+ − − − − Conclusion:lacIsisdominanttolacI+,andcanblockenzymeproduc8on fromanyloca8on Thepointofgene8csandgenemapping -Makepredic8onsaboutcrossessoonecanconstruct organismswithdesiredtraits -Oneuseofconstructedorganismsistogenerate modelsandhypothesesabouthowtheywork -Genera8ngamodelfromgene8cdataocenrequires imagina(onandcrea(vity -notjustanapplica8onofpurelogic... thisisanart(butanartrootedindata) JacobandMonod'smodelforthelacoperon Repressorproteinblocks transcrip8onby bindinglacODNA DNA O I Z Y DNA A Inducer X mRNA Repressor protein mRNA β-galactosidase permease transacetylase InducerblocksrepressorbindingtolacO, allowingtranscrip8onoflacZYAgenes lacP,"thepromoter"iswhereRNA polymeraseini8atestranscrip8on structuralgenes lacO lacOnon-func8onal muta8onsmaphere lacZ lacY lacA lacOcons8tu8ve(Oc) muta8onsmaphere "promoter" lacP lacO lacZ "operator" lacY lacA JacobandMonod'smodelforthelacoperon x Lacrepressorbindingtotheoperator(O)blocks RNApolymerasefrommakingmRNA Muta8onsinpromoter(lacP)thatcan'tbind RNApolymerasecannotmakeanylacenzymes Muta8onsinoperator(lacOc)thatcan'tbind repressormakelacenzymescons<tu<vely Norepressor(lacI)atoperator(lacO)à RNApolymeraseunblockedàgenesac8ve Inducer(IPTG)inhibitsrepressorbindingà RNApolymeraseunblockedàgenesac8ve Norepressor(lacI-): cannotblockRNApolymeraseàcons8tu8veexpression Muta8ons(lacOc)inoperatorregionthatareunabletobind repressor: cannotblockRNApolymeraseàcons8tu8veexpression lacIisdominanttolacI-,andfunc8onsintrans I- X P O Z Y P O Z Y Noac8ve repressor I Norepressorresultsinenzymesproducedcons8tu8vely Func8onallacIproteinsencodedonanF'episomecanrepress transcrip8onofenzymegenesonbothcopies lacIsisdominanttolacI+,andfunc8onsintrans Is P O Z Y I P O Z Y lacIscannotbindinducer,soits repressionac8vitycan'tturnoff Isrepressorcannotbindinducer NormallacIrepressorprotein repressestranscrip8on. Inducerblocksthisrepression, allowingtranscrip8on. lacIsrepressorbindslacOsites inbothchromosomeandFepisome,blockingtranscrip8on. lacOallelesactincisandaffectonlyneighboring enzymegenes I I P O Z Y Repressor Expressionblockedin cannotbind mutantoperator absenceofinducer P Oc Z Y Expressionallowedintheabsenceofinducer ModeltestedwiththePaJaMaexperiment (ArthurPArdee,FrancoisJAcob,JacquesMOnod) Hfr(I+Z+)matedtoF-I-Z-strainintheabsenceof inducer,whilemonitoringβ-galac8vity -Hfrtakes1hrtotransfertheIandZgenes Ini8ally,expectnoβ-galactosidaseac8vity HfrdonorshavelacZ,butnoinducer,sostaysoff F-recipientsarelacZ- can'tproduceenzymewithorwithoutinducer PaJaMaexperiment-Hfr(I+Z+)matedtoF-I-Z- Acer1hr,theI+Z+genesentertherecipientF-cells lacZ+isnowintherecipient(I-Z-)cell Norepressorà nothingtoblockthenewlacZgeneà β-galactosidaseproduc8onexpectedtobegin β-galproduc8onexpectedtoeventuallyshutdown donorI+geneonthetransferredDNAtakes 8metomakeenoughrepressortoshutdown produc8onofβ-gal Anyobservedac8vityfromalready-madeβ-gal PaJaMaexperiment-Hfr(I+Z+)matedtoF-I-Z- I- X P Noac8ve repressor F' I+ non-func<onalβ-galproduced allthe8mewithoutrepressor O Z- Y- Onceenoughrepressoris made,newβ-galproduc8on stops P Later:repressorsynthesized fromtheF'lacI+copybuilds upandrepressestranscrip8on O Z+ Justacertransfer: norepressor, β-galcanbemade Y+ PaJaMaexperiment β-galmade following ma8ng Acerawhile,nonewβ-galmade; noincreaseinac8vity Timeacerma8ng(h) PaJaMaexperiment-Hfr(I+Z+)matedtoF-I-Z- I- X P Noac8ve repressor F' I+ non-func<onalβ-galproduced allthe8mewithoutrepressor O Z- Y- Onceenoughrepressoris made,newβ-galproduc8on stops P Later:repressorsynthesized fromtheF'lacI+copybuilds upandrepressestranscrip8on O Z+ Justacertransfer: norepressor, β-galcanbemade Whathappensifweaddinducer? β-gallevelsexpectedtorise Y+ PaJaMaexperiment–withinducer Timeacerma8ng(h) lacrepressorisaninducer-regulatedDNA bindingprotein lacrepressorisatetramericproteinthatbindsIPTG Intheabsenceofinducer,bindsbindslacoperatorvery 8ghtly(K=1013M-1)butveryweaklytootherDNAsequences Bindsfasterthandiffusionrateconstantinsolu8on repressorslidesalongDNAquicklyun8litfindsthetarget operatorsequence TightbindingtooperatorpreventsRNApolymerasebinding Thebasesequenceofthelacoperator OCmuta8ons blockrepressor binding LacRepressortetramerboundtolacoperatorDNA Twooperator sequencesbound Q:Whereisthelactose? protein-DNA contacts= lacOCmutated posi8ons Tetrameriza8on region CataboliterepressionandthediauxicshiE • GlucoseisthecarbonsourceofchoiceforE.coli • Ifalterna8vessuchaslactose,arabinose,orgalactoseare presentinhighconcentra8ons,theydon'tgetmetabolized un<ltheglucoseisusedup(diauxicshic) • Cataboliterepression-preventsthewastefulproduc8onof energyandmaterialcostlyenzymes. Youdoasimilarthing:youusuallyeatyourfavorite foodfirst,beforeswitchingtolesstastyfoods Thekine8csoflacoperonmRNAsynthesisfollowingitsinduc8onwith IPTG,andofitsdegrada8onacerglucoseaddi8on. Diauxicshic Cataboliterepression isviaadenylatecyclase andcAMP inhibitedindirectlyby highglucoseconcentra8ons Withglucose:low[cAMP] ATP adenylatecyclase cAMP Noglucose:high,steady[cAMP] breaksdowncAMP tomaintainasteady concentra8on phophodiesterase AsiteinthepromoterisboundbyaCAP-cAMP complexthatrecruitsRNApolymerase cAMP inac,veCAP (cataboliteac,vatorprotein) ac,veCAP-cAMP(boundtocAMP) lacoperon CAP-cAMPbindstotheCAPsiteinthe promoter,facilita8ngbindingof RNApolymerase RNApolymerase Transcrip8onini8ated mRNA transcrip8on andtransla8on Molecularanatomyofthelacpromoter CAP RNApolymerase lacrepressor(lacI)at operator(lacO) atpromoter Shine-Dalgarno= ribosomebindingsite inmRNA Note:"promoter"isocenusedtorefernotjusttotheregionwhere RNApolymerasebinds,butalsototheen8restretchofregulatoryDNA sequencesneartheRNApolymerasebindingsite,includingtheoperator Posi8veandnega8veregula8onatthelacpromoter Highglucose:lowcAMPà CAPdoesn'tbinditssiteà RNApolymeraseNOTrecruited Result:nolacmRNA Nolactose:repressorbindsoperatorà RNApolymeraseblocked Posi8veandnega8veregula8onatthelacpromoter Highglucose:lowcAMPà CAPdoesn'tbinditssiteà RNApolymeraseNOTrecruited Result:li*lelacmRNA Lactose:repressorbindslactoseà doesNOTbindoperatorà doesNOTblockRNApolymerase Posi8veandnega8veregula8onatthelacpromoter (corrected) Lowglucose:highcAMPà CAPbindsitssiteà RNApolymeraserecruited Lactose:repressorbindslactoseà doesNOTbindoperatorà doesNOTblockRNApolymerase Result:abundantlacmRNA Next8me: RNApolymeraseinprokaryotes Moreexamplesofprokaryotegeneregula8on Generegula8oninelephantsand othereukaryotes Now:GradeQuiz3 5'-ACCTGCTACGG-3' 3'-TGGACGATGCC-5' 5'-CCGTAGCAGGT-3' DNA polymerase synthesizes leading strand continuously Parental DNA 5ʹ 3ʹ 3ʹ 5ʹ DNA polymerase starts DNA synthesis at 3ʹ end of primer, continues in 5ʹ → 3ʹ direction 5ʹ Origin of replication Lagging strand synthesized in short Okazaki fragments, later joined by DNA ligase 3ʹ 5ʹ aminoacyltRNAsynthetasesa*achthe correctaminoacidtotheencodingtRNA No3'OH,no furtherextension; thechainstops simulatestheliver toxinprocessing Ifthechemicalisamutagen, onewillgeneratemanysecondary muta8onsthatcompensatefor theoriginalhis-muta8on, allowinggrowthonlow-hismedia smallribosome subunit Streptomycininteracts with16SRNAand S12protein (encodedbyrpsLgene) MutH MutL Non-homologous endjoining(NHEJ) Homologous recombina8on Met 3ʹ U A C 5ʹ 5ʹ A U G 3ʹ Pi Initiator tRNA + GTP GDP mRNA 5ʹ Start codon mRNA binding site (Shine-Dalgarno seq) 3ʹ Small ribosomal subunit 5ʹ Individualswithxeroderma pigmentosum,muta8onsin UVrepairpathwayenzymes areveryfreckled,sensi8veto sunlight,andusuallydieof skincancerbyage20 C A B C C D GCcontent:moreGCà higherTm Sugar Adenine(A) Sugar Thymine(T) Sugar Sugar Guanine(G) Cytosine(C) GChas3hydrogen bonds;AThas2 Meselson-Stahlexperiment Semi-conserva8ve DNAreplica8on Duetoredundancyinthegene8ccode,somemuta8ons (silentorsynonymous)donotaffecttheproteinsequence