Introduction to Operon - Lac operon.pdf

Operon
The concept of how gene regulation occurs at the transcription level in bacteria was provided by the classical model called the operon model
Proposed by Jacob and Monod in 1961 (Noble prize in 1965 in Physiology & Medicine).
What is operon?
An operon is a genetic regulatory system mostly seen in prokaryotes and bacteriophages in which a group of structural genes are transcribed together under the control of a single promoter.

1. Ms. M. Arthi, M.Sc., M.Phil., NET., SET.,
Assistant Professor
Department of Microbiology
DKM College for Women (A), Vellore

2. •Atypicalbacterialcellcontainsseveralthousandgenes.
•Somegenescarryoutuniversaltasksandareconstantlyactive,called
housekeepinggenes/constitutivegenes.
•Eg.Theenzymesthatareinvolvedinthecentralmetabolicpathwaysare
producedalmostallofthetimebecausetheycatalyzereactionsinthecellthat
areneededroutinely.
•Themajorityoftheothergenes,becomeactiveonlywhentheirbyproducts
areneeded.
•Suchgeneexpressioniscontrolledinaccordancewiththeneedofthecell.
•Regulationofgeneexpressionoccursduringtranscriptioninitiation.
•Prokaryotictranscriptionalregulationisaccomplishedbygeneregulatory
proteinsthatbindwithregulatorysequencesnearthetranscriptionstartsite.

3. •Regulatoryproteins,theproductsofregulatorygenes,areoftwotypes
activatorandrepressor
•Absenceofbothactivatorandrepressor-Basalleveloftranscription
occurs.
•Bindingofarepressor-decreasestranscription(lessthanthebasallevel).
•Bindingofanactivator-increasestranscription(abovethebasallevel),
•Presenceofbothfunctionalactivatorandrepressor-theactionofthe
repressorovercomestheactivator.

4. •Theconceptofhowgeneregulationoccursatthe
transcriptionlevelinbacteriawasprovidedby
theclassicalmodelcalledtheoperonmodel
•ProposedbyJacobandMonodin1961(Noble
prizein1965inPhysiology&Medicine).
Whatisoperon?
•Anoperonisageneticregulatorysystemmostly
seeninprokaryotesandbacteriophagesinwhich
agroupofstructuralgenesaretranscribed
togetherunderthecontrolofasinglepromoter.
•Also found in some eukaryotes.
•The main difference is
•Expression of prokaryotic operons –produce
polycistronic mRNAs
•Expression of eukaryotic operons –produce
monocistronicmRNAs

5. Regulatory genes
•Promoter
•Operator
Structural Genes
➢Operon can be defined as a stretch of DNA consisting of structural genes and a regulatory genes
Promoter:nucleotidesequencerecognizedbytheRNA
polymerase,whereitbindsandtranscribesthestructural
genes.
Operator:asegmentofDNAactasasiteforrepressor
binding,whichblocksthetranscription.Presentb/wthe
promoterandthestructuralgenes.Itoverlapswiththe
promotersequences.
Structuralgenes:Thesegenesarecodingsequences,
transcribedintoapolycistronicmRNAbytheRNAPolymerase,
whichfurthertranslatedintoindividualproteins.

7. Regulatory genes
control the expression of the structural genes based on the cells requirement.
•non-coding
DNA,
•regulates the
transcription of
neighbouring
genes.
•Eg.Promoter &
operator
Cis-acting
regulatory
genes
Cis-acting
regulatory
genes
•region of DNA,
which usually
codes for a
diffusible
protein, which
can bind and
regulate the
transcription of
distant genes.
•Eg.repressor &
activator.
Trans-
acting
regulatory
genes
Trans-
acting
regulatory
genes

9. Regulation of an operon
two types of transcriptional control
•Repressorisanegativeregulatory
molecule,bindstotheoperator
geneandinterfereswiththe
expressionofgenes.
•Activatorisapositiveregulatory
molecule,enhances/inducesthe
expressionofthegenes.
Negative
regulation
Positive
regulation

10. Operons can also be either inducible or repressible
Inducible
operon
Transcription is normally off, it
needs to be induced or turned
on
Negative inducible
Positive inducible
Eg: Lactose / Lac operon
Repressible
operon
Transcription is normally on, it
needs to be repressed or turned
off
Negative repressible
Positive repressible
Eg: Tryptophan / Trpoperon

11. a) Negative inducible operon
•Theregulatorygeneencodesarepressorwhichbindstotheoperator,blocksthebindingofRNApolymerase
tothepromoterandpreventsthetranscription.Normalsituation–OFF.
•Fortheinitiationoftranscription,therepressorhastoberemovedfromtheoperatorsite.Theactionofthe
repressorproteinisblockedbythebindingofaninducer.TranscriptionisturnedON–induced.

12. b) Positive inducible operon
•Theregulatorygeneencodesaactivatorproteinwhichisinactive,itcannotactivatethetranscription.Normalsituation–OFF.
•Fortheinitiationoftranscription,aninducerbindstotheinactiveactivatorproteinandconvertittoanactiveproteinwhich
bindstotheactivator-bindingsite(upstreamofthepromoter).BindingoftheactivatortoitsregulatoryregionpromotestheRNA
polymerasebinding.TranscriptionisturnedON–induced.

13. c) Negative repressible operon
•Theregulatorygenesynthesizesaninactiverepressorcalledaporepressorwhichcannotbindstoanoperator,soRNApolymerase
readilybindstothepromoterandproceedsthetranscriptionandtranscribethestructuralgenes.Normalsituation–ON.
•Toturnoffthetranscription,theaporepressorhastobeactivated.Asmallmoleculecalledcorepressorbindstotheaporepressor
andmakesitactiverepressorprotein,whichbindstooperator.TranscriptionisturnedOFF–repressed.

14. d) Positive repressible operon
•Theregulatorygeneencodesanactivatorproteinwhichisactive,itreadilybindstotheoperatorand
activatethetranscription.Normalsituation–ON.
•Transcriptionisinhibited,aninhibitorbindstotheactiveactivatorproteinandconvertittoaninactive
proteinandpreventsitsbindingtotheoperator.TranscriptionisturnedOFF–repressed.

15. Inducible
Repressible
Positive control
Negative control
•Normally OFF
•Turned ON
•Normally ON
•Turned OFF
•Regulatory protein
act as an Activator
•Regulatory protein
act as a Repressor

16. Classic examples of operon
Lactose or Lac operon
Tryptophan or Trpoperon
Arabinose or Ara operon

17. Lac operon

18. Lactose (Lac) Operon
•LacoperonisanexampleofInducibleoperon(NormallyOFF,IthastoswitchedON–inducedby
aninducer-allolactose).
•LacoperonispresentinE.coli,abacteriacommonlyfoundinthehumanintestines.
•Itenablesthebacteriatometabolizelactoseasthesourceofenergy.Lactoseismetabolizedwiththe
helpofβ-galactosidaseenzyme.
Inducer
The operon is
expressed only when
lactose is present and
glucose is absent.

19. Structure of the lac operon
Structural
Genes
Three Structural genes: lacZ, lacY,
and lacA.
These genes are transcribed from a single promoter
as a single polycistronic mRNA.
Regulatory
Genes
The operon is regulated both
negativelyand positively.
Negative control is brought about by the lac repressor,
which is the product of the lacIgene. The operator is the
site of lac repressor binding.
Positive control results from the action of CAP. CAP binds the
CAP site located just upstream from the lac promoter. CAP is,
responsible for catabolite repression.

20. 1. In the absence of Lactose
•The lactose-metabolizing enzymes are not required.
•The lac repressor protein binds with operator and blocks the RNA polymerase binding
and stops the transcription.

21. 2. In the presence of Lactose
•The inducer -Allolactose binds to the repressor, results in conformational change of the
repressor, no longer binds to the operator.
•RNA pol can bind to the promoter and proceeds the transcription.

22. The effect of availability of glucose:
•TheE.colicanutilizelactose,howeverglucoseispreferredsourceofenergyforthecell,as
theutilizationoflactoseisenergeticallymoreexpensivethanglucose.
•Hence,lactoseisutilizedbythecell,onlywhentheglucoseispresentinlowlevelor
absent.
•lacoperonisturnedononlywhenglucoseisunavailableanditisturnedoffinpresence
oftheglucose(eveniflactoseispresent).
•Thelacoperonregulationdependsontheavailabilityofglucose.

23. Weak promotor
•Lacpromoterisaweakpromoter
•TheRNApolymerasecannotbindtothepromotermoreefficientlyonitsown.
•Cataboliteactivatorprotein(CAP)helpstheRNAPolymerasebinding.
•CAPproteinbindstoCataboliteActivatorProtein(CAP)bindingsiteisapositiveregulatory
sitelocatedjustupstreamofthelacoperonpromoter,
•TheCAPisadimerprotein,whichhasbindingsitesforcAMPandDNA.
•WhencAMPbindsCAP,itsaffinityfortheDNAincreases.
•cAMPlevelsareonlywhenglucoseisabsent,withoutcAMPtheCAPisinactive.
•WhenboundtoDNA,CAP-cAMPpromotestranscriptionbyaidingRNApolymerasebindtothe
promotermoreefficiently.
CAP
cAMP
DNA Binding Site

24. 3. When Lactose is present; glucose is absent
•ThepositiveregulatoryeffectofCAP-CAPhelpstocontrolthelacOperondependingonthe
concentrationofglucose.
•Whenglucoseisabsent,cAMPbindsCAP,whichthenbindsCAPbindingsite.cAMPboundCAP
anchorstheRNApolymerasetothepromoterandinitiatedthetranscriptionoflacoperongenes.

25. 4. When Lactose is present; glucose is present
•whenglucoseispresent,theamountofavailablecAMPdecreases.
•withoutcAMP,CAPcannotbindDNA,whichinturncausesweakbindingofRNApolymeraseto
thelacoperonpromoter
•Asaresultonlyfewtranscriptsareproducedandlactoseutilizingenzymesarenotefficiently
produced.
•lacoperonisturnedononlywhen‘glucose‘thatisthepreferredsourceofenergyisunavailable
andlactoseispresent.

26. Condition Lactose Glucose CAP binding Repressor binding
Level of
Transcription
1
•Present
•Inducer-
Allolactose is
present
•Absent
•cAMP level is
high
•CAP binds with cAMP
•CAP-cAMP complex helps the
RNA Polymerase to bind
strongly with the promoter
•Allolactose binds to
repressor
•Inactive repressor
cannot binds to the
operator
High level of
transcription
2
•Present
•Inducer-
Allolactose is
present
•Present
•cAMP level is
low
•CAP cannot binds with cAMP,
as its level is low
•RNA Polymerase cannot bind
strongly with the promoter
•Allolactose binds to
repressor
•Inactive repressor
cannot binds to the
operator
Low level of
transcription
3
•Absent
•No Allolactose
•Present
•cAMP level is
low
•No CAP binding
•Repressor binds to the
operator and blocks the
binding of RNA
polymerase
No transcription
4
•Absent
•No Allolactose
•Absent
•cAMP level is
high
•CAP binds with cAMP
•CAP-cAMP complex helps the
RNA Polymerase to bind
strongly with the promoter
•Repressor binds to the
operator and blocks the
binding of RNA
polymerase
No transcription