Download CHAPTER 13 – PROKARYOTE GENES: E. COLI LAC OPERON

PROKARYOTEGENES:E.COLILACOPERON–CHAPTER13
CHAPTER13–PROKARYOTEGENES:E.COLILACOPERON
Figure1.
Electron micrograph of growing E. coli.
Someshowtheconstrictionatthelocation
where daughter cells separate. The
colouringisfalse.
(Flickr-NIAID-CCBY2.0)
INTRODUCTION
Withmostorganisms,everycellcontainsessentially
the same genomic sequence. How then do cells
develop and function differently from each other?
The answer lies in the regulation of gene
expression. Only a subset of all the genes is
expressed(i.e.arefunctionallyactive)inanygiven
cell participating in a particular biological process.
Geneexpressionisregulatedatmanydifferentsteps
along the process that converts DNA information
into active proteins. In the first stage, transcript
abundancecanbecontrolledbyregulatingtherate
oftranscriptioninitiationandprocessing,aswellas
the degradation of transcripts. In many cases,
higher abundance of a gene’s transcripts is
correlated with its increased expression. We will
focusontranscriptionalregulationinE.coli(Figure
1).Beaware,however,thatcellsalsoregulatethe
overallactivityofgenesinotherways.Forexample,
by controlling the rate of mRNA translation,
processing, and degradation, as well as the posttranslational modification of proteins and protein
complexes.
OPENGENETICSLECTURES–FALL2015
1. THELACOPERON–AMODELPROKARYOTE
GENE
Early insights into mechanisms of transcriptional
regulation came from studies of E. coli by
researchers Francois Jacob & Jacques Monod (see
Section 4 on page 4). In E. coli, and many other
bacteria, genes encoding several different
polypeptidesmaybelocatedinasingletranscription
unitcalledanoperon.Thegenesinanoperonshare
the same transcriptional regulation, but are
translated individually into separate polypeptides.
Most prokaryote genes are not organized as
operons, but are transcribed as single polypeptide
units.
Eukaryotesdonotgroupgenestogetherasoperons
(anexceptionisC.elegansandafewotherspecies).
1.1. BASICLACOPERONSTRUCTURE
E. coli encounters many different sugars in its
environment. These sugars, such as lactose and
glucose, require different enzymes for their
metabolism. Three of the enzymes for lactose
metabolismaregroupedinthelacoperon:lacZ,
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CHAPTER13–PROKARYOTEGENES:E.COLILACOPERON
lacY,andlacA(Figure2).LacZencodesanenzyme
calledβ-galactosidase,whichdigestslactoseintoits
twoconstituentsugars:glucoseandgalactose.lacY
isapermeasethathelpstotransferlactoseintothe
Figure2.
DiagramofasegmentofanE.colichromosomecontaining
thelacoperon,aswellasthelacIcodingregion.Thevarious
genes and cis-elements are not drawn to scale. (OriginalDeyholos-CCBY-NC3.0)
cell.Finally,lacAisatrans-acetylase;therelevance
ofwhichinlactosemetabolismisnotentirelyclear.
Transcriptionofthelacoperonnormallyoccursonly
when lactose is available for it to digest.
Presumably, this avoids wasting energy in the
synthesis of enzymes for which no substrate is
present.Inthelacoperon,thereisasinglemRNA
transcript that includes coding sequences for all
threeenzymesandiscalledapolycistronicmRNA.A
cistroninthiscontextisequivalenttoagene.
1.2. CIS-ANDTRANS-REGULATORS
Inadditiontothesethreeprotein-codinggenes,the
lac operon contains several short DNA sequences
that do not encode proteins, but instead act as
bindingsitesforproteinsinvolvedintranscriptional
regulationoftheoperon.Inthelacoperon,these
sequences are called P (promoter), O (operator),
and CBS (CAP-binding site). Collectively, sequence
elements such as these are called cis-elements
because they must be located adjacently to the
samepieceofDNAinordertoperformcorrectly.On
the other hand, elements outside from the target
DNA (such as the proteins that bind to these ciselements) are called trans-regulators because (as
diffusiblemolecules)theydonotnecessarilyneedto
beencodedonthesamepieceofDNAasthegenes
theyregulate.
2. NEGATIVEREGULATION–INDUCERSAND
REPRESSORS
2.1. lacIENCODESANALLOSTERICALLYREGULATED
REPRESSOR Oneofthemajortrans-regulatorsofthelacoperon
is encoded by lacI, a gene located just upstream
from the lac operon (Figure 2). Four identical
molecules of lacI proteins assemble together to
formahomotetramercalledarepressor(Figure3).
Thisrepressoristrans-actingandbindstotwocisactingoperatorsequencesadjacenttothepromoter
ofthelacoperon.Bindingoftherepressorprevents
RNA polymerase from binding to the promoter
(Figure 2, Figure 5.). Therefore, the operon is not
transcribed when the operator sequence is
occupiedbyarepressor.
Figure3.
StructureoflacIhomotetramerboundtoDNA.
(Original-Deyholos-CCBY-NC3.0)
2.2. THEREPRESSORALSOBINDSLACTOSE
(ALLOLACTOSE)
Besides its ability to bind to specific DNA sequences at the
attheoperator,anotherimportantpropertyofthelacIprotein
lacI protein is its ability to bind to allolactose. If lactose is
lactoseispresent,β-galactosidase(β-gal)enzymesconverta
convertafewofthelactosemoleculesintoallolactose(
Figure4.).
This allolactose can then bind to the lacI protein.
This alters the shape of the protein in a way that
preventsitfrombindingtotheoperator.Proteins
whichchangetheirshapeandfunctionalproperties
after binding to a ligand are said to be regulated
throughanallostericmechanism.
Therefore, in the presence of lactose (which β-gal
converts to allolatose), the repressor doesn’t bind
theoperatorsequenceandthusRNApolymeraseis
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PROKARYOTEGENES:E.COLILACOPERON–CHAPTER13
3. POSITIVEREGULATION–CAP,CAMP&
POLYMERASE
Figure4.
β-galactosidase catalyzes two reactions: one converts
lactosetoallolactose;theothersplitslactoseintogalactose
andglucose.(Original-Nickle-CCBY-NC4.0)
abletobindtothepromoterandtranscribethelac
operon.Thisleadstoamoderatelevelofexpression
of the mRNA encoding the lacZ, lacY, and lacA
genes.Thiskindofsecondarymoleculethatbindsto
either activator or repressor and induces the
productionofspecificenzymeiscalledaninducer.
The role of lacI in regulating the lac operon is
summarizedinFigure5.
A second aspect of lac operon regulation is
conferred by a trans-acting factor called cAMP
binding protein (CAP, Figure 6). CAP is another
example of an allosterically regulated trans-factor.
Only when the CAP protein is bound to cAMP can
another part of the protein bind to a specific ciselement within the lac promoter called the CAP
bindingsequence(CBS).CBSisimmediatelyinfront
ofthepromoter(P),andthusisacis-actingelement.
WhenCAPisboundtoattheCBS,RNApolymerase
is better able to bind to the promoter and initiate
transcription. Thus, the presence of cAMP
ultimatelyleadstoafurtherincreaseinlacoperon
transcription.
Figure6.
CAP, when bound to cAMP, increases RNApol binding to
the lac operon promoter. cAMP is produced only when
glucose[Glc]islow.(Original-Deyholos-CCBY-NC3.0)
Figure5.
When the concentration of lactose [Lac] is low, lacI
tetramersbindtooperatorsequences(O),therebyblocking
binding of RNApol (green) to the promoter (P).
Alternatively, when [Lac] is high, lactose binds to lacI,
preventingtherepressorfrombindingtoO,andallowing
transcriptionbyRNApol.
(Original-Deyholos-CCBY-NC3.0)
OPENGENETICSLECTURES–FALL2015
ThephysiologicalsignificanceofregulationbycAMP
becomes more obvious in the context of the
followinginformation.TheconcentrationofcAMPis
inverselyproportionaltotheabundanceofglucose.
When glucose concentrations are low, an enzyme
called adenylate cyclase is able to produce cAMP
from ATP. Evidently, E. coli prefers glucose over
lactose, and so expresses the lac operon at high
levels only when glucose is absent and lactose is
present. This provides another layer of adaptive
control of lac operon expression: only in the
presenceoflactoseandintheabsenceofglucoseis
theoperonexpressedatitshighestlevels.
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CHAPTER13–PROKARYOTEGENES:E.COLILACOPERON
4. THEUSEOFMUTANTSTOSTUDYTHELAC
OPERON
4.1. SINGLEMUTANTSOFTHELACOPERON
The lac operon and its regulators were first
characterized by studying mutants of E. coli that
exhibited various abnormalities in lactose
metabolism.MutationscanoccurinanyofthelacZ,
lacY, and lacA genes. Such mutations result in
altered protein sequences, and cause nonfunctional products. These are mutations in the
proteincodingsequences(non-regulatory).
Other mutants can cause the lac operon to be
expressed constitutively, meaning the operon was
transcribed whether or not lactose was present in
themedium.Rememberthatnormallytheoperonis
onlytranscribediflactoseispresent.Suchmutants
are called constitutive mutants. Constitutive
mutants are always on and are unregulated by
inducers.TheseincludelacOandlacIgenes.
4.2. OPERATORMUTATIONS
The operator locus (lacO) - One example is O-, in
whichamutationinanoperatorsequencereduces
or precludes the repressor (the lacI gene product)
from recognizing and binding to the operator
sequence.Thus,inO-mutants,lacZ,lacY,andlacA
are expressed whether or not lactose is present.
Notethatthismutationiscisdominant(onlyaffects
the genes on the same chromosome) but not in
trans(otherDNAmolecule).Anothercommonname
for a defective operator is Oc to refer to its
constitutiveexpression.O-andOcaresynonyms.
NotethatconstitutivelyexpressedO-mutantsmay
notbemaximallyexpressed,andtheextentofthe
mutation can also affect the level of expression.
(Table1)
c
Table1.ConstitutivelyexpressedO mutantsmaynotbe
maximallyexpressedandhavevariouslevelsofexpression.
Level
Genotype
100%
-
c
lacI O norepressor
10-20%
+
c
lacI O repressorfailstobindtightly
~1%
P O ,high
glucose
0%
P orZ +
c
-
-
Explanation
basaltranscription,constitutive
notranscription
4.3. INDUCERMUTATIONS(lacILOCUS)
ThelacIlocushastwotypesofmutations:I-andIS.
Figure7.
+ + - + +
+ - + - +
BothE.colistrandswithgenotypesI O Z Y A /FI O Z Y A
S + + + +
- - + + +
and I O Z Y A /F I O Z Y A will induce all the lac genes
becauserepressorcannotbindtotheO sequenceonthe
F-factorandcannotpreventtranscription.
(Original-Locke-CCBY-NC3.0)
PAGE4
OneclassofmutantalleleforlacI(calledI-)either(1)
preventstheproductionofarepressorpolypeptide,
or (2) produces a polypeptide that cannot bind to
theoperatorsequence.Notethatthesetwoalleles
would have different genetic sequences, but the
phenotype is the same. Theoretically, we should
havebetterlocusnamestoproperlyidentifyspecific
alleles, but for now we’ll group them under one
name. With this form of mutation, the repressor
cannotbindandtranscriptioncanoccurwithoutthe
presence of inducer (allolactose). This can also be
referredtoasa“constitutiveexpresser”ofthelac
operon: the absence of repressor binding permits
transcription.
NotethatI+isdominantoverI-.Forexample,inE.
colistrainwithI+Z-Y+A+/FI-Z+Y-A+, thelacgeneswill
not be inducible because the I+ allele will still
OPENGENETICSLECTURES–FALL2015
producefunctionalrepressorsthatbindtooperator
sequence,preventingtranscription.(Figure8)
PROKARYOTEGENES:E.COLILACOPERON–CHAPTER13
I+ and I- in trans. Therefore, E. coli strains with the
genotypes 1) ISZ+Y+A+/F I+Z+Y-A+ and 2) ISZ+Y+A+/F IZ+Y+A+, the lac Z, lac Y and lac A genes will not be
inducible(Figure9).
TherepressorproteinencodedbylacIgenehasat
least two independent functional domains. This is
the reason why it different mutations can give
differentclassesofmutantphenotypes.(Figure10)
Figure8.
+ - + +
- + - +
E. coli strain with genotype I Z Y A /F I Z Y A will not
producelacZ,lacYandlacAproducts.
(Original-Locke-CCBY-NC3.0)
TheotherclassofmutantallelesforlacIarecalled
Is. The altered amino sequence of their proteins
remove the “allosteric site”. This means the
repressorpolypeptidecannotbindallolactose–and
therefore it cannot change its shape. Even in the
presence of lactose, it remains attached to the
operator,sothelacZ,lacYandlacAgenescannot
beexpressed(noRNApolymerase,noprotein).This
mutant constitutively represses the lac operon
whetherlactoseispresentornot.Thelacoperonis
not expressed at all and this mutant is called a
“super-repressor”.Is isthereforedominanttoboth
Figure9.
S + + +
+ + - +
E. coli strains with genotypes 1) I Z Y A /F I Z Y A and 2)
S + + +
- + + +
I Z Y A /F I Z Y A will not produce lac Z, lac Y and lac A
products.(Original-Locke-CCBY-NC3.0)
OPENGENETICSLECTURES–FALL2015
Figure10.
Because the repressor encoded by lac I gene has
independent domains, mutations can also occur
independently.
(Original-Deyholos/Locke-CCBY-NC3.0)
4.4. THEF-FACTORANDTWOlacOPERONSINASINGLE
CELL –PARTIALDIPLOIDIN E.COLI Morecanbelearnedabouttheregulationofthelac
operonwhentwodifferentcopies(eachcontaining
mutations) are present in one cell. This can be
accomplishedbyusinganF-factor(alsoknownasa
plasmid)tocarryonecopy,whiletheotherisonthe
genomicE.colichromosome.Thisresultsinapartial
diploid in E. coli that contains two independent
copies(alleles)ofthelacoperonandlacI.
AnF-factor(namedsobecauseitcreates“fertility”
inthecellwhichcontainsit)isanepisome.Thisisa
self-replicating extrachromosomal piece of DNA: it
is outside of the large, circular bacterial
chromosome but has its own origin of replication.
JacobandMonodexploredtheregulationofthelac
operon by introducing different genotypes into
bacterialcellsandnotinghowtheyrespondedinthe
presenceofabsenceofsugars,namelyglucoseand
lactoseusingthemutantclassesdiscussedabove.
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CHAPTER13–PROKARYOTEGENES:E.COLILACOPERON
Forexample,thegenotypeofahostbacteriumthat
has a lacI- gene that is supplied with F factor
containinglacI+canbewrittenaslacI-/F+lacI+.Infact,
it doesn’t matter which piece of DNA (genomic or
episome)haswhichoperon,sowecanleaveoffthe
“F”altogether–equivalentnotationislacI-/lacI+.
whichbindstoCAPprotein.CAPproteincanbindto
DNA and increase the level of transcription. High
glucoselevelhaltsadenylatecyclasefromproducing
cAMPfromATP.Hence,cAMPwillnotbindtoCAP
proteinandinturn,CAPwillnotbindtoDNAandthe
leveloftranscriptionwouldbelow.
Thus,cellswithtwocopiesofagenesequenceare
partial diploids, called merozygotes (Figure 11).
Theseallowresearcherstotesttheregulationwith
differentcombinationsofmutationsinonecell.For
example,theF-factorcopymayhaveaISmutation
whilethegenomiccopymighthaveanOCmutation.
How would this cell respond to the
presence/absence of lactose (or glucose)? This
partial diploid can be used to determine that IS is
dominanttoI+,whichinturnisdominanttoI-.Itcan
alsobeusedtoshowtheOCmutationonlyactsin
cis-whilethelacImutationcanactintrans-.
Innegativeregulation,therepressorproteinactsto
preventtranscription.Inducerbindstorepressorto
alter conformation so it no longer binds to the
operatorsequenceandtranscriptioncantakeplace.
Highlevelsoflactose(inducer)wouldallosterically
inhibit repressor and therefore would not prevent
transcription.Lowlevelsoflactosewouldnotcause
inhibition to the repressor, so transcription would
beprevented.Variousformsofregulationinthelac
operonarefoundinFigure12.
Figure11.
Adiagramshowingamerozygote.Thisisaformofbacterialcell
s
whereaplasmid(blue)hasanormaloperonbutalacI allelefor
the repressor. Not all bacterial cells have plasmids. The
chromosome(red)hasanormalrepressorandtheoperonhasa
lacY allele. The super-repressor will inactivate both operons
becauseitwillbindtobothoperators(atrans-actingeffect).Note
thatboththeplasmidandchromosomehavetheirownoriginsof
replication.(Original-Nickle-CCBY-NC4.0)
5. SUMMARY
Inpositiveregulation,lowlevelsofglucose(inducer)
allowadenylatecyclasetoproducecAMPfromATP,
Figure12.
PAGE6
Whenglucose[Glc]andlactose[Lac]arebothhigh,thelac
operonistranscribedatamoderatelevel,becauseCAP(in
theabsenceofcAMP)isunabletobindtoitscorresponding
cis-element(yellow)andthereforecannothelptostabilize
binding of RNApol at the promoter. Alternatively, when
[Glc]islow,and[Lac]ishigh,CAPandcAMPcanbindnear
thepromoterandincreasefurtherthetranscriptionofthe
lacoperon.(Original-Deyholos-CCBY-NC3.0)
OPENGENETICSLECTURES–FALL2015
PROKARYOTEGENES:E.COLILACOPERON–CHAPTER13
___________________________________________________________________________
SUMMARY:
•
Regulationofgeneexpressionisessentialtothenormaldevelopmentandefficientfunctioning
ofcells
•
Geneexpressionmayberegulatedbymanymechanisms,includingthoseaffectingtranscript
abundance,proteinabundance,andpost-translationalmodifications
•
Regulationoftranscriptabundancemayinvolvecontrollingtherateofinitiationandelongation
oftranscription,aswellastranscriptsplicing,stability,andturnover
•
The rate of initiation of transcription is related to the presence of RNA polymerase and
associatedproteinsatthepromoter.
•
RNApolmaybeblockedfromthepromoterbyrepressors,ormayberecruitedorstabilizedat
thepromoterbyotherproteinsincludingtranscriptionfactors
•
Thelacoperonisaclassic,fundamentalparadigmdemonstratingbothpositiveandnegative
regulationthroughallostericeffectsontrans-factors.
KEYTERMS:
geneexpression
transcriptionalregulation
operon
lactose
glucose
lacoperon
lacZ
lacY
lacA
β-galactosidase
permease
trans-acetylase
P/promoter
O/operator
CBS
CAP-bindingsite
cis-elements
trans-regulators
OPENGENETICSLECTURES–FALL2015
lacI
homotetramer
repressor
inducer
allosteric
cAMPbindingprotein
CAP
CAPbindingsequence
CBS
adenylatecyclase
constitutive
Oc/I-/Is
cisdominant
cis-actingfactors
transdominant
trans-actingfactors
F-factor/episome
merozygotes
PAGE7
CHAPTER13–PROKARYOTEGENES:E.COLILACOPERON
STUDYQUESTIONS:
1) Listallpointsinthe“centraldogma”ofgene
actionthemechanismsthatcanbeusedto
regulategeneexpression.
2) With respect to the expression of βgalactosidase,whatwouldbethephenotype
ofeachofthefollowingstrainsofE.coli?
3) In the E. coli strains listed below, some genes
arepresentonboththechromosome,andthe
extrachromosomal F-factor episome. The
genotypesofthechromosomeandepisomeare
separated by a slash. What will be the βgalactosidasephenotypeofthesestrains?Allof
the strains are grown in media that lacks
glucose.
Usethesymbols:
+++ Lotsofβ-galactosidaseactivity(100%)
+ Moderateβ-galactosidaseactivity(10-20%)
- Noβ-galactosidaseactivity(0-1%)
a)
b)
c)
d)
e)
f)
g)
h)
i)
j)
k)
l)
m)
n)
o)
p)
q)
r)
s)
+
+
+
Usethesymbols:
+++ Lotsofβ-galactosidaseactivity(100%)
+ Moderateβ-galactosidaseactivity(10-20%)
- Noβ-galactosidaseactivity(0-1%)
+
I ,O ,Z ,Y (noglucose,nolactose)
+
+ + +
I ,O ,Z ,Y (noglucose,highlactose)
+
+ + +
I ,O ,Z ,Y (highglucose,nolactose)
+
+ + +
I ,O ,Z ,Y (highglucose,highlactose)
+
+ - +
I ,O ,Z ,Y (noglucose,nolactose)
+
+ - +
I ,O ,Z ,Y (highglucose,highlactose)
+
+ + I ,O ,Z ,Y (highglucose,highlactose)
+
c + +
I ,O ,Z ,Y (noglucose,nolactose)
+
c + +
I ,O ,Z ,Y (noglucose,highlactose)
+
c + +
I ,O ,Z ,Y (highglucose,nolactose)
+
c + +
I ,O ,Z ,Y (highglucose,highlactose)
+ + +
I ,O ,Z ,Y (noglucose,nolactose)
+ + +
I ,O ,Z ,Y (noglucose,highlactose)
+ + +
I ,O ,Z ,Y (highglucose,nolactose)
+ + +
I ,O ,Z ,Y (highglucose,highlactose)
s
+ + +
I ,O ,Z ,Y (noglucose,nolactose)
s
+ + +
I ,O ,Z ,Y (noglucose,highlactose)
s
+ + +
I ,O ,Z ,Y (highglucose,nolactose)
s
+ + +
I ,O ,Z ,Y (highglucose,highlactose)
a)
b)
c)
d)
e)
f)
g)
h)
i)
j)
k)
l)
m)
n)
o)
p)
I+,O+,Z+,Y+/I+,O-,Z-,Y-(highlactose)
I+,O+,Z+,Y+/I+,O-,Z-,Y-(nolactose)
I+,O+,Z-,Y+/I+,O-,Z+,Y+(highlactose)
I+,O+,Z-,Y+/I+,O-,Z+,Y+(nolactose)
I+,O+,Z-,Y+/I-,O+,Z+,Y+(highlactose)
I+,O+,Z-,Y+/I-,O+,Z+,Y+(nolactose)
I-,O+,Z+,Y+/I+,O+,Z-,Y+(highlactose)
I-,O+,Z+,Y+/I+,O+,Z-,Y+(nolactose)
I+,Oc,Z+,Y+/I+,O+,Z-,Y+(highlactose)
I+,Oc,Z+,Y+/I+,O+,Z-,Y+(nolactose)
I+,O+,Z-,Y+/I+,Oc,Z+,Y+(highlactose)
I+,O+,Z-,Y+/I+,Oc,Z+,Y+(nolactose)
I+,O+,Z-,Y+/Is,O+,Z+,Y+(highlactose)
I+,O+,Z-,Y+/Is,O+,Z+,Y+(nolactose)
Is,O+,Z+,Y+/I+,O+,Z-,Y+(highlactose)
Is,O+,Z+,Y+/I+,O+,Z-,Y+(nolactose)
4) WhatgenotypesofE.coliwouldbemostuseful
indemonstratingthatthelacOoperatorisacisactingregulatoryfactor?
5) What genotypes of E. coli would be useful in
demonstratingthatthelacIrepressorisatransactingregulatoryfactor?
6) Whatwouldbetheeffectofthefollowinglossof-functionmutationsontheexpressionofthe
lacoperon?
a) loss-of-functionofadenylatecyclase
b) lossofDNAbindingabilityofCAP
c) lossofcAMPbindingabilityofCAP
d) mutationofCAPbindingsite(CBS)
cis-elementsothatCAPcouldnotbind
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OPENGENETICSLECTURES–FALL2015