prokaryote genes coli lac operon chapter...

PROKARYOTEGENES:E.COLILACOPERON–CHAPTER13

OPENGENETICSLECTURES–FALL2015 PAGE1

CHAPTER13–PROKARYOTEGENES:E.COLILACOPERON

Figure1.Electron micrograph of growing E. coli.Someshowtheconstrictionatthelocationwhere daughter cells separate. Thecolouringisfalse.(Flickr-NIAID-CCBY2.0)

INTRODUCTIONWithmostorganisms,everycellcontainsessentiallythe same genomic sequence. How then do cellsdevelopand functiondifferently fromeachother?The answer lies in the regulation of geneexpression. Only a subset of all the genes isexpressed(i.e.arefunctionallyactive) inanygivencell participating in a particular biological process.Geneexpressionisregulatedatmanydifferentstepsalong the process that converts DNA informationinto active proteins. In the first stage, transcriptabundancecanbecontrolledbyregulatingtherateoftranscriptioninitiationandprocessing,aswellasthe degradation of transcripts. In many cases,higher abundance of a gene’s transcripts iscorrelated with its increased expression. WewillfocusontranscriptionalregulationinE.coli(Figure1).Beaware,however,thatcellsalsoregulatetheoverallactivityofgenesinotherways.Forexample,by controlling the rate of mRNA translation,processing, and degradation, as well as the post-translational modification of proteins and proteincomplexes.

1. THELACOPERON–AMODELPROKARYOTEGENE

Early insights into mechanisms of transcriptionalregulation came from studies of E. coli byresearchers Francois Jacob& JacquesMonod (seeSection 4 on page 4). In E. coli, andmany otherbacteria, genes encoding several differentpolypeptidesmaybelocatedinasingletranscriptionunitcalledanoperon.Thegenesinanoperonsharethe same transcriptional regulation, but aretranslated individually into separate polypeptides.Most prokaryote genes are not organized asoperons,butare transcribedas singlepolypeptideunits.

Eukaryotesdonotgroupgenestogetherasoperons(anexceptionisC.elegansandafewotherspecies).

1.1. BASICLACOPERONSTRUCTUREE. coli encounters many different sugars in itsenvironment. These sugars, such as lactose andglucose, require different enzymes for theirmetabolism. Three of the enzymes for lactosemetabolismaregroupedinthelacoperon:lacZ,


PAGE2 OPENGENETICSLECTURES–FALL2015

lacY,andlacA(Figure2).LacZencodesanenzymecalledβ-galactosidase,whichdigestslactoseintoitstwoconstituentsugars:glucoseandgalactose.lacYisapermeasethathelpstotransferlactoseintothe

cell.Finally,lacAisatrans-acetylase;therelevanceofwhichinlactosemetabolismisnotentirelyclear.Transcriptionofthelacoperonnormallyoccursonlywhen lactose is available for it to digest.Presumably, this avoids wasting energy in thesynthesis of enzymes for which no substrate ispresent.Inthelacoperon,thereisasinglemRNAtranscript that includes coding sequences for allthreeenzymesandiscalledapolycistronicmRNA.Acistroninthiscontextisequivalenttoagene.

1.2. CIS-ANDTRANS-REGULATORSInadditiontothesethreeprotein-codinggenes,thelac operon contains several short DNA sequencesthat do not encode proteins, but instead act asbindingsitesforproteinsinvolvedintranscriptionalregulationoftheoperon.Inthe lacoperon,thesesequences are called P (promoter),O (operator),andCBS (CAP-binding site).Collectively, sequenceelements such as these are called cis-elementsbecause they must be located adjacently to thesamepieceofDNAinordertoperformcorrectly.Onthe other hand, elements outside from the targetDNA (such as the proteins that bind to these cis-elements) are called trans-regulators because (asdiffusiblemolecules)theydonotnecessarilyneedtobeencodedonthesamepieceofDNAasthegenestheyregulate.

2. NEGATIVEREGULATION–INDUCERSANDREPRESSORS

2.1. lacIENCODESANALLOSTERICALLYREGULATEDREPRESSOR

Oneofthemajortrans-regulatorsofthelacoperonis encoded by lacI, a gene located just upstreamfrom the lac operon (Figure 2). Four identicalmolecules of lacI proteins assemble together toformahomotetramercalledarepressor(Figure3).Thisrepressoristrans-actingandbindstotwocis-actingoperatorsequencesadjacenttothepromoterofthelacoperon.BindingoftherepressorpreventsRNA polymerase from binding to the promoter(Figure 2,Figure 5.). Therefore, the operon is nottranscribed when the operator sequence isoccupiedbyarepressor.

Figure3.StructureoflacIhomotetramerboundtoDNA.(Original-Deyholos-CCBY-NC3.0)

2.2. THEREPRESSORALSOBINDSLACTOSE(ALLOLACTOSE)

Besides its ability to bind to specific DNA sequences at theattheoperator,anotherimportantpropertyofthelacIproteinlacI protein is its ability to bind to allolactose. If lactose islactose ispresent,β-galactosidase(β-gal)enzymesconvertaconvertafewofthelactosemoleculesintoallolactose(

Figure4.).

This allolactose can then bind to the lacI protein.This alters the shape of the protein in away thatpreventsitfrombindingtotheoperator. Proteinswhichchangetheirshapeandfunctionalpropertiesafter binding to a ligand are said to be regulatedthroughanallostericmechanism.

Therefore, in thepresenceof lactose (whichβ-galconverts to allolatose), the repressordoesn’t bindtheoperatorsequenceandthusRNApolymeraseis

Figure2.DiagramofasegmentofanE.colichromosomecontainingthelacoperon,aswellasthelacIcodingregion.Thevariousgenes and cis-elements are not drawn to scale. (Original-Deyholos-CCBY-NC3.0)



abletobindtothepromoterandtranscribethelacoperon.Thisleadstoamoderatelevelofexpressionof the mRNA encoding the lacZ, lacY, and lacAgenes.Thiskindofsecondarymoleculethatbindstoeither activator or repressor and induces theproductionofspecificenzymeiscalledan inducer.The role of lacI in regulating the lac operon issummarizedinFigure5.

Figure5.When the concentration of lactose [Lac] is low, lacItetramersbindtooperatorsequences(O),therebyblockingbinding of RNApol (green) to the promoter (P).Alternatively, when [Lac] is high, lactose binds to lacI,preventingtherepressorfrombindingtoO,andallowingtranscriptionbyRNApol.(Original-Deyholos-CCBY-NC3.0)

3. POSITIVEREGULATION–CAP,CAMP&POLYMERASE

A second aspect of lac operon regulation isconferred by a trans-acting factor called cAMPbinding protein (CAP, Figure 6). CAP is anotherexampleofanallosterically regulated trans-factor.Onlywhen theCAPprotein isbound tocAMPcananother part of the protein bind to a specific cis-element within the lac promoter called the CAPbindingsequence(CBS).CBSisimmediatelyinfrontofthepromoter(P),andthusisacis-actingelement.WhenCAPisboundtoattheCBS,RNApolymeraseisbetterable tobindto thepromoterand initiatetranscription. Thus, the presence of cAMPultimatelyleadstoafurtherincreaseinlacoperontranscription.

ThephysiologicalsignificanceofregulationbycAMPbecomes more obvious in the context of thefollowinginformation.TheconcentrationofcAMPisinverselyproportionaltotheabundanceofglucose.When glucose concentrations are low, an enzymecalledadenylate cyclase is able to produce cAMPfrom ATP. Evidently, E. coli prefers glucose overlactose, and so expresses the lac operon at highlevels only when glucose is absent and lactose ispresent. This provides another layer of adaptivecontrol of lac operon expression: only in thepresenceoflactoseandintheabsenceofglucoseistheoperonexpressedatitshighestlevels.

Figure6.CAP,whenbound to cAMP, increasesRNApolbinding tothe lac operon promoter. cAMP is produced only whenglucose[Glc]islow.(Original-Deyholos-CCBY-NC3.0)

Figure4.β-galactosidase catalyzes two reactions: one convertslactosetoallolactose;theothersplitslactoseintogalactoseandglucose.(Original-Nickle-CCBY-NC4.0)



4. THEUSEOFMUTANTSTOSTUDYTHELACOPERON

4.1. SINGLEMUTANTSOFTHELACOPERONThe lac operon and its regulators were firstcharacterized by studying mutants of E. coli thatexhibited various abnormalities in lactosemetabolism.MutationscanoccurinanyofthelacZ,lacY, and lacA genes. Such mutations result inaltered protein sequences, and cause non-functional products. These are mutations in theproteincodingsequences(non-regulatory).

Other mutants can cause the lac operon to beexpressed constitutively,meaning theoperonwastranscribedwhetherornot lactosewaspresent inthemedium.Rememberthatnormallytheoperonisonlytranscribediflactoseispresent.Suchmutantsare called constitutive mutants. Constitutivemutants are always on and are unregulated byinducers.TheseincludelacOandlacIgenes.

4.2. OPERATORMUTATIONSThe operator locus (lacO) - One example isO-, inwhichamutationinanoperatorsequencereduces

orprecludes the repressor (the lacI geneproduct)from recognizing and binding to the operatorsequence.Thus,inO-mutants, lacZ, lacY,and lacAare expressed whether or not lactose is present.Notethatthismutationiscisdominant(onlyaffectsthe genes on the same chromosome) but not intrans(otherDNAmolecule).Anothercommonnamefor a defective operator is Oc to refer to itsconstitutiveexpression.O-andOcaresynonyms.

NotethatconstitutivelyexpressedO-mutantsmaynotbemaximallyexpressed,andtheextentofthemutation can also affect the level of expression.(Table1)

4.3. INDUCERMUTATIONS(lacILOCUS)

ThelacIlocushastwotypesofmutations:I-andIS.

OneclassofmutantalleleforlacI(calledI-)either(1)preventstheproductionofarepressorpolypeptide,or (2)producesapolypeptide that cannotbind totheoperatorsequence.Notethatthesetwoalleleswould have different genetic sequences, but thephenotype is the same. Theoretically, we shouldhavebetterlocusnamestoproperlyidentifyspecificalleles, but for now we’ll group them under onename. With this form of mutation, the repressorcannotbindandtranscriptioncanoccurwithoutthepresenceof inducer (allolactose). This can also bereferredtoasa“constitutiveexpresser”ofthe lacoperon: the absence of repressor binding permitstranscription.

Notethat I+ isdominantover I-.Forexample, inE.colistrainwithI+Z-Y+A+/FI-Z+Y-A+,the lacgeneswillnot be inducible because the I+ allele will still

Figure7.BothE.colistrandswithgenotypesI+O+Z-Y+A+/FI+O-Z+Y-A+

and ISO+Z+Y+A+/F I-O-Z+Y+A+ will induce all the lac genesbecauserepressorcannotbindtotheO-sequenceontheF-factorandcannotpreventtranscription.(Original-Locke-CCBY-NC3.0)

Table1.ConstitutivelyexpressedOcmutantsmaynotbemaximallyexpressedandhavevariouslevelsofexpression.Level Genotype Explanation100% lacI-Oc norepressor

10-20% lacI+Oc repressorfailstobindtightly

~1% P+Oc,highglucose basaltranscription,constitutive

0% P-orZ- notranscription



producefunctionalrepressorsthatbindtooperatorsequence,preventingtranscription.(Figure8)

Theotherclassofmutantallelesfor lacIarecalledIs. The altered amino sequence of their proteinsremove the “allosteric site”. This means therepressorpolypeptidecannotbindallolactose–andtherefore it cannot change its shape. Even in thepresence of lactose, it remains attached to theoperator,sothelacZ,lacYandlacAgenescannotbeexpressed(noRNApolymerase,noprotein).Thismutant constitutively represses the lac operonwhetherlactoseispresentornot.Thelacoperonisnot expressed at all and this mutant is called a“super-repressor”.Isisthereforedominanttoboth

I+and I- in trans.Therefore,E.coli strainswith thegenotypes1) ISZ+Y+A+/F I+Z+Y-A+and2) ISZ+Y+A+/F I-

Z+Y+A+,the lacZ, lacY and lacA geneswillnotbeinducible(Figure9).

Therepressorproteinencodedby lacIgenehasatleast two independent functional domains. This isthe reason why it different mutations can givedifferentclassesofmutantphenotypes.(Figure10)

4.4. THEF-FACTORANDTWOlacOPERONSINASINGLECELL–PARTIALDIPLOIDINE.COLI

Morecanbelearnedabouttheregulationofthelacoperonwhentwodifferentcopies(eachcontainingmutations) are present in one cell. This can beaccomplishedbyusinganF-factor(alsoknownasaplasmid)tocarryonecopy,whiletheotherisonthegenomicE.colichromosome.Thisresultsinapartialdiploid in E. coli that contains two independentcopies(alleles)ofthelacoperonandlacI.

AnF-factor(namedsobecauseitcreates“fertility”inthecellwhichcontainsit)isanepisome.Thisisaself-replicatingextrachromosomalpieceofDNA: itis outside of the large, circular bacterialchromosomebuthas itsownoriginof replication.JacobandMonodexploredtheregulationofthelacoperon by introducing different genotypes intobacterialcellsandnotinghowtheyrespondedinthepresenceofabsenceofsugars,namelyglucoseandlactoseusingthemutantclassesdiscussedabove.

Figure8.E. coli strain with genotype I+Z-Y+A+/F I-Z+Y-A+ will notproducelacZ,lacYandlacAproducts.(Original-Locke-CCBY-NC3.0)

Figure9.E. coli strainswith genotypes 1) ISZ+Y+A+/F I+Z+Y-A+ and 2)ISZ+Y+A+/F I-Z+Y+A+ will not produce lac Z, lac Y and lac Aproducts.(Original-Locke-CCBY-NC3.0)

Figure10.Because the repressor encoded by lac I gene hasindependent domains, mutations can also occurindependently.(Original-Deyholos/Locke-CCBY-NC3.0)



Forexample,thegenotypeofahostbacteriumthathas a lacI- gene that is supplied with F factorcontaininglacI+canbewrittenaslacI-/F+lacI+.Infact,itdoesn’tmatterwhichpieceofDNA (genomicorepisome)haswhichoperon,sowecanleaveoffthe“F”altogether–equivalentnotationislacI-/lacI+.

Thus,cellswithtwocopiesofagenesequencearepartial diploids, called merozygotes (Figure 11).Theseallowresearcherstotesttheregulationwithdifferentcombinationsofmutationsinonecell.Forexample,theF-factorcopymayhaveaISmutationwhilethegenomiccopymighthaveanOCmutation.How would this cell respond to thepresence/absence of lactose (or glucose)? Thispartial diploid can be used to determine that IS isdominanttoI+,whichinturnisdominanttoI-.ItcanalsobeusedtoshowtheOCmutationonlyacts incis-whilethelacImutationcanactintrans-.

Figure11.Adiagramshowingamerozygote.Thisisaformofbacterialcellwhereaplasmid(blue)hasanormaloperonbuta lacIsalleleforthe repressor. Not all bacterial cells have plasmids. Thechromosome(red)hasanormalrepressorandtheoperonhasalacY- allele. The super-repressor will inactivate both operonsbecauseitwillbindtobothoperators(atrans-actingeffect).Notethatboththeplasmidandchromosomehavetheirownoriginsofreplication.(Original-Nickle-CCBY-NC4.0)

5. SUMMARYInpositiveregulation,lowlevelsofglucose(inducer)allowadenylatecyclasetoproducecAMPfromATP,

whichbindstoCAPprotein.CAPproteincanbindtoDNA and increase the level of transcription.HighglucoselevelhaltsadenylatecyclasefromproducingcAMPfromATP.Hence,cAMPwillnotbindtoCAPproteinandinturn,CAPwillnotbindtoDNAandtheleveloftranscriptionwouldbelow.

Innegativeregulation,therepressorproteinactstopreventtranscription.Inducerbindstorepressortoalter conformation so it no longer binds to theoperatorsequenceandtranscriptioncantakeplace.High levelsof lactose(inducer)wouldallostericallyinhibit repressorand thereforewouldnotpreventtranscription.Lowlevelsoflactosewouldnotcauseinhibition to the repressor, so transcriptionwouldbeprevented.VariousformsofregulationinthelacoperonarefoundinFigure12.

Figure12.Whenglucose[Glc]andlactose[Lac]arebothhigh,thelacoperonistranscribedatamoderatelevel,becauseCAP(intheabsenceofcAMP)isunabletobindtoitscorrespondingcis-element(yellow)andthereforecannothelptostabilizebindingofRNApol at thepromoter. Alternatively,when[Glc]islow,and[Lac]ishigh,CAPandcAMPcanbindnearthepromoterandincreasefurtherthetranscriptionofthelacoperon.(Original-Deyholos-CCBY-NC3.0)



___________________________________________________________________________SUMMARY:

• Regulationofgeneexpressionisessentialtothenormaldevelopmentandefficientfunctioningofcells

• Geneexpressionmayberegulatedbymanymechanisms,includingthoseaffectingtranscriptabundance,proteinabundance,andpost-translationalmodifications

• Regulationoftranscriptabundancemayinvolvecontrollingtherateofinitiationandelongationoftranscription,aswellastranscriptsplicing,stability,andturnover

• The rate of initiation of transcription is related to the presence of RNA polymerase andassociatedproteinsatthepromoter.

• RNApolmaybeblockedfromthepromoterbyrepressors,ormayberecruitedorstabilizedatthepromoterbyotherproteinsincludingtranscriptionfactors

• The lacoperonisaclassic,fundamentalparadigmdemonstratingbothpositiveandnegativeregulationthroughallostericeffectsontrans-factors.

KEYTERMS:geneexpressiontranscriptionalregulationoperonlactoseglucoselacoperonlacZlacYlacAβ-galactosidasepermeasetrans-acetylaseP/promoterO/operatorCBSCAP-bindingsitecis-elementstrans-regulators

lacIhomotetramerrepressorinducerallostericcAMPbindingproteinCAPCAPbindingsequenceCBSadenylatecyclaseconstitutiveOc/I-/Is

cisdominantcis-actingfactorstransdominanttrans-actingfactorsF-factor/episomemerozygotes



STUDYQUESTIONS:1) Listallpointsinthe“centraldogma”ofgene

actionthemechanismsthatcanbeusedtoregulategeneexpression.

2) With respect to the expression of β-galactosidase,whatwouldbethephenotypeofeachofthefollowingstrainsofE.coli?

Usethesymbols:+++ Lotsofβ-galactosidaseactivity(100%) + Moderateβ-galactosidaseactivity(10-20%) - Noβ-galactosidaseactivity(0-1%)a) I+,O+,Z+,Y+(noglucose,nolactose) b) I+,O+,Z+,Y+(noglucose,highlactose)c) I+,O+,Z+,Y+(highglucose,nolactose)d) I+,O+,Z+,Y+(highglucose,highlactose)e) I+,O+,Z-,Y+(noglucose,nolactose)f) I+,O+,Z-,Y+(highglucose,highlactose)g) I+,O+,Z+,Y-(highglucose,highlactose)h) I+,Oc,Z+,Y+(noglucose,nolactose)i) I+,Oc,Z+,Y+(noglucose,highlactose)j) I+,Oc,Z+,Y+(highglucose,nolactose)k) I+,Oc,Z+,Y+(highglucose,highlactose)l) I-,O+,Z+,Y+(noglucose,nolactose)m) I-,O+,Z+,Y+(noglucose,highlactose)n) I-,O+,Z+,Y+(highglucose,nolactose)o) I-,O+,Z+,Y+(highglucose,highlactose)p) Is,O+,Z+,Y+(noglucose,nolactose)q) Is,O+,Z+,Y+(noglucose,highlactose)r) Is,O+,Z+,Y+(highglucose,nolactose)s) Is,O+,Z+,Y+(highglucose,highlactose)

3) In theE. coli strains listedbelow, somegenesarepresentonboththechromosome,andtheextrachromosomal F-factor episome. Thegenotypesofthechromosomeandepisomeareseparated by a slash. What will be the β-

galactosidasephenotypeofthesestrains?Allofthe strains are grown in media that lacksglucose.Usethesymbols:+++ Lotsofβ-galactosidaseactivity(100%) + Moderateβ-galactosidaseactivity(10-20%) - Noβ-galactosidaseactivity(0-1%)a) I+,O+,Z+,Y+/I+,O-,Z-,Y-(highlactose)b) I+,O+,Z+,Y+/I+,O-,Z-,Y-(nolactose)c) I+,O+,Z-,Y+/I+,O-,Z+,Y+(highlactose)d) I+,O+,Z-,Y+/I+,O-,Z+,Y+(nolactose)e) I+,O+,Z-,Y+/I-,O+,Z+,Y+(highlactose)f) I+,O+,Z-,Y+/I-,O+,Z+,Y+(nolactose)g) I-,O+,Z+,Y+/I+,O+,Z-,Y+(highlactose)h) I-,O+,Z+,Y+/I+,O+,Z-,Y+(nolactose)i) I+,Oc,Z+,Y+/I+,O+,Z-,Y+(highlactose)j) I+,Oc,Z+,Y+/I+,O+,Z-,Y+(nolactose)k) I+,O+,Z-,Y+/I+,Oc,Z+,Y+(highlactose)l) I+,O+,Z-,Y+/I+,Oc,Z+,Y+(nolactose)m) I+,O+,Z-,Y+/Is,O+,Z+,Y+(highlactose)n) I+,O+,Z-,Y+/Is,O+,Z+,Y+(nolactose)o) Is,O+,Z+,Y+/I+,O+,Z-,Y+(highlactose)p) Is,O+,Z+,Y+/I+,O+,Z-,Y+(nolactose)

4) WhatgenotypesofE.coliwouldbemostuseful

indemonstratingthatthelacOoperatorisacis-actingregulatoryfactor?

5) What genotypes of E. coli would be useful indemonstratingthatthelacIrepressorisatrans-actingregulatoryfactor?

6) Whatwouldbetheeffectofthefollowingloss-of-functionmutationsontheexpressionofthelacoperon?a) loss-of-functionofadenylatecyclaseb) lossofDNAbindingabilityofCAPc) lossofcAMPbindingabilityofCAPd) mutationofCAPbindingsite(CBS)

cis-elementsothatCAPcouldnotbind

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