Download inducers - Navin Pokala

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