Prokaryotic Gene RegulationProkaryotic Gene Regulation

Coordinate regulation of genes Coordinate regulation of genes involved in similar functionsinvolved in similar functions

Types of Control Types of Control

Negative ControlNegative Control Product of regulatory gene Product of regulatory gene inhibits transcriptioninhibits transcription

Positive ControlPositive Control Product of regulatory gene Product of regulatory gene enhances transcription enhances transcription

OperonOperon

• Unit of coordinate gene expressionUnit of coordinate gene expression

• Includes structural genes and their Includes structural genes and their adjacent regulatory elementsadjacent regulatory elements

• We will considerWe will consider– Lac operon (inducible)Lac operon (inducible)– Ara operon (inducible)Ara operon (inducible)– Trp operon (repressible)Trp operon (repressible)

Types of Operons Types of Operons

InducibleInducible Initial condition: OFFInitial condition: OFF

Inducer switches operon ONInducer switches operon ON

Repressible Repressible Initial condition: ON Initial condition: ON

Repressor switches operon OFFRepressor switches operon OFF

Lac Z Lac Y Lac AP OPi IPcrp crp

Regulation of the Lac OperonRegulation of the Lac Operon

DNA FunctionDNA Function Protein FunctionProtein Function

PPcrpcrpPromoter for crp genePromoter for crp gene

crpcrp Gene for CAP proteinGene for CAP protein Positive regulatorPositive regulator

PPiiPromoter for I genePromoter for I gene

II Gene for Lac RepressorGene for Lac Repressor Negative regulatorNegative regulator

PP Promoter for Structural GenesPromoter for Structural Genes

OO OperatorOperator

Lac ZLac Z Gene for B-galactosidaseGene for B-galactosidase Cleaves lactoseCleaves lactose

Lac YLac Y Gene for PermeaseGene for Permease Lactose transportLactose transport

Lac ALac A Gene for AcetylaseGene for Acetylase UnknownUnknown

Str

uctu

ral G

enes

Str

uctu

ral G

enes

Lac Z Lac Y Lac AP OPi IPcrp crp

Transcription from the Lac OperonTranscription from the Lac Operon

RNA polymerase binds to the promoter and RNA polymerase binds to the promoter and produces a polycistronic mRNA from the produces a polycistronic mRNA from the Lac Z, Y and A genes. All three proteins areLac Z, Y and A genes. All three proteins areproduced.produced.

Pol

Z, Y, A mRNAZ, Y, A mRNA

TranscriptionTranscription

B-galactosidaseB-galactosidase

TranslationTranslation

PermeasePermease AcetylaseAcetylase

Lac Z Lac Y Lac AP OPi IPcrp crp

Regulation of the Lac Operon: Low lactose,Regulation of the Lac Operon: Low lactose, High glucose High glucose

Transcription from PTranscription from Pcrpcrp and P and Pii is constitutive: is constitutive:

always expressed in an unregulated fashion.always expressed in an unregulated fashion.Active repressor binds to operator and preventsActive repressor binds to operator and preventsRNA polymerase from reaching structural genes.RNA polymerase from reaching structural genes.

Active Active repressorrepressor

crp mRNAcrp mRNA I mRNAI mRNA

Inactive Inactive CAP proteinCAP protein

No mRNA producedNo mRNA produced

No Z, Y, A proteins producedNo Z, Y, A proteins produced

Pol

Regulation of the Lac Operon: High lactose,Regulation of the Lac Operon: High lactose, High glucose High glucose

Lac Z Lac Y Lac AP OPi IPcrp crp

Active Active repressorrepressor

crp mRNAcrp mRNA I mRNAI mRNA

Inactive Inactive CAP proteinCAP protein

Lactose

Lactose (inducer) binds to the repressor and Lactose (inducer) binds to the repressor and inactivates it. RNA polymerase transcribes inactivates it. RNA polymerase transcribes Lac Z, Y and A at low frequency.Lac Z, Y and A at low frequency.

+

Inactive Inactive repressorrepressor

Pol

Z, Y, A mRNAZ, Y, A mRNA

TranscriptionTranscription

TranslationTranslation

B-galactosidaseB-galactosidase PermeasePermease AcetylaseAcetylase

Regulation of the Lac Operon: High lactose,Regulation of the Lac Operon: High lactose, Low glucose Low glucose

TranslationTranslation

B-galactosidaseB-galactosidase PermeasePermease AcetylaseAcetylase

Lac Z Lac Y Lac AP OPi IPcrp crp

Active Active repressorrepressor

crp mRNAcrp mRNA I mRNAI mRNA

Inactive Inactive CAP proteinCAP protein

Lactose+

Inactive Inactive repressorrepressor

+

cAMP is produced when glucose levels are low. cAMP cAMP is produced when glucose levels are low. cAMP activates CAP. Active CAP binds to the promoter to activates CAP. Active CAP binds to the promoter to increase RNA polymerase binding. RNA polymerase increase RNA polymerase binding. RNA polymerase transcribes Lac Z, Y and A at HIGH frequency.transcribes Lac Z, Y and A at HIGH frequency.

cAMP

Active Active CAP proteinCAP protein

Pol

Z, Y, A mRNAZ, Y, A mRNA

TranscriptionTranscription

Regulation of the Lac Operon: Low lactose,Regulation of the Lac Operon: Low lactose, Low glucose Low glucose

Active Active repressorrepressor

Lac Z Lac Y Lac AP OPi IPcrp crp

crp mRNAcrp mRNA I mRNAI mRNA

Inactive Inactive CAP proteinCAP protein

cAMP

+

Active Active CAP proteinCAP protein

Although RNA polymerase binding is enhanced by Although RNA polymerase binding is enhanced by Active CAP, the operator is blocked by active repressor.Active CAP, the operator is blocked by active repressor.RNA polymerase cannot transcribe Z, Y and A. RNA polymerase cannot transcribe Z, Y and A.

No mRNA producedNo mRNA produced

No Z, Y, A proteins producedNo Z, Y, A proteins produced

Pol

CAP Protein Structure Allows Binding to DNACAP Protein Structure Allows Binding to DNA

• Domains are regions on a protein with specific Domains are regions on a protein with specific functions; motifs are characteristic structures functions; motifs are characteristic structures within a domainwithin a domain

• CAP has a DNA binding domain with a helix-CAP has a DNA binding domain with a helix-turn-helix structural motifturn-helix structural motif

• Helices fit into the major groove on DNAHelices fit into the major groove on DNA

Summary of Lac Operon Summary of Lac Operon Regulation Regulation

Level of Level of LactoseLactose

Level of Level of GlucoseGlucose

Lac OperonLac Operon

LowLow HighHigh

LowLow LowLow

HighHigh HighHigh

HighHigh LowLow

OffOff

OffOff

On at low frequencyOn at low frequency

On at high frequencyOn at high frequency

Lac Z Lac Y Lac AP OPi IPcrp CRP

Mutations of the Lac OperonMutations of the Lac Operon

II++ Functional RepressorFunctional Repressor Trans-actingTrans-acting

II-- Non-functional RepressorNon-functional Repressor Trans-actingTrans-acting

IIss Superrepressor Superrepressor

(cannot bind lactose)(cannot bind lactose)

Trans-actingTrans-acting

IIss> I> I++> I> I--

Functional genes: IFunctional genes: I+ + PP+ + O O+ + ZZ+ + YY+ + AA++

The diffusible product of the IThe diffusible product of the I++ or I or ISS allele can allele can associate with an operator on the same piece of associate with an operator on the same piece of DNA (cis) or on a separate piece of DNA (trans).DNA (cis) or on a separate piece of DNA (trans).

Lac Z Lac Y Lac AP OPi IPcrp CRP

Mutations of the Lac OperonMutations of the Lac Operon

PP++ Functional PromoterFunctional Promoter Cis-actingCis-acting

PP-- Non-functional PromoterNon-functional Promoter Cis-actingCis-acting

OO++ Functional OperatorFunctional Operator Cis-actingCis-acting

OOcc Non-functional OperatorNon-functional Operator

(Operator Constitutive)(Operator Constitutive)

Cis-actingCis-acting

OO-- Non-functional OperatorNon-functional Operator

(Operator region deleted)(Operator region deleted)

Cis-actingCis-acting

Functional genes: IFunctional genes: I+ + PP+ + O O+ + ZZ+ + YY+ + AA++

Lac Z Lac Y Lac AP OPi IPcrp CRP

Mutations of the Lac OperonMutations of the Lac Operon

ZZ++ Functional B-galactosidase Functional B-galactosidase

ZZ-- Non-functional gene for B-galactosidaseNon-functional gene for B-galactosidase

YY++ Functional PermeaseFunctional Permease

YY-- Non-functional gene for PermeaseNon-functional gene for Permease

AA++ Functional AcetylaseFunctional Acetylase

AA-- Non-functional gene for AcetylaseNon-functional gene for Acetylase

Functional genes: IFunctional genes: I+ + PP+ + O O+ + ZZ+ + YY+ + AA++

A mutation in one structural gene does not affect A mutation in one structural gene does not affect the production of proteins from the other the production of proteins from the other structural genes. structural genes.

Lac Operon Mutations, Page 3-24 Lac Operon Mutations, Page 3-24

II++PP++OO++ZZ++YY++

II--PP++OO++ZZ++YY++

II++PP--OO++ZZ++YY++

II++PP++OOccZZ++YY++

II++PP++OO++ZZ--YY++/ I/ I--PP++OO++ZZ++YY++

II++PP++OOccZZ++YY--/ I/ I++PP++OO++ZZ--YY++

II++PP++OOccZZ--YY++/ I/ I--PP++OO++ZZ++YY--

IIssPP++OO++ZZ++YY--/ I/ I--PP++OO++ZZ--YY++

II++PP--OOccZZ++YY++/ I/ I++PP++OO++ZZ--YY--

No lactoseNo lactose LactoseLactose LactoseLactoseNo lactoseNo lactose

B-GalactosidaseB-Galactosidase PermeasePermease

-- + -- +-- + -- +

+ + + ++ + + +

-- -- -- ---- -- -- --

+ + + ++ + + +

-- + -- +-- + -- + + + -- ++ + -- +

-- + + +-- + + +

-- -- -- ---- -- -- --

-- -- -- ---- -- -- --

DNA FunctionDNA Function Protein FunctionProtein Function

Ara CAra C Codes for C proteinCodes for C protein Positive and Positive and Negative regulatorNegative regulator

II Initiator (promoter region)Initiator (promoter region) Binds C proteinBinds C protein

OO OperatorOperator Binds C proteinBinds C protein

Ara BAra B Structural geneStructural gene

Ara AAra A Structural geneStructural gene

Ara DAra D Structural geneStructural gene

Arabinose OperonArabinose Operon

C Protein Exerts Positive and C Protein Exerts Positive and Negative Control of the Ara OperonNegative Control of the Ara Operon

Arabinose present

Arabinose absent

Summary of Ara Operon RegulationSummary of Ara Operon Regulation Level of Level of ArabinoseArabinose

Level of Level of GlucoseGlucose

Ara OperonAra Operon

LowLow HighHigh

LowLow LowLow

HighHigh HighHigh

HighHigh LowLow

OffOffC protein bound to O and I,C protein bound to O and I,Inhibiting transcriptionInhibiting transcription

OffOffC protein bound to O and IC protein bound to O and I

On at low frequencyOn at low frequencyC protein + arabinose bound C protein + arabinose bound to I, enhancing transcriptionto I, enhancing transcription

On at high frequencyOn at high frequencyC protein + arabinose bound C protein + arabinose bound to I and cAMP + CAP boundto I and cAMP + CAP boundto I, enhancing transcriptionto I, enhancing transcription

DNA FunctionDNA Function RNA/Protein FunctionRNA/Protein Function

Trp RTrp R Gene for Gene for repressorrepressor

Binds to operator to inhibit Binds to operator to inhibit transcriptiontranscription

PP PromoterPromoter

OO OperatorOperator

Trp E, D, Trp E, D, C, B, AC, B, A

Structural Structural genesgenes

Enzymes acting in pathway to Enzymes acting in pathway to produce tryptophan.produce tryptophan.

Gene order correlates with Gene order correlates with order of reactions in pathway.order of reactions in pathway.

5’ UTR5’ UTR(Leader)(Leader)

Premature termination of Premature termination of transcription when trp levels are transcription when trp levels are highhigh

Tryptophan OperonTryptophan Operon

trpA

Control of Trp Operon TranscriptionControl of Trp Operon TranscriptionTrp Repressor is Inactive Trp Repressor is Inactive Initial State: ON Initial State: ON

Trp binding activates Repressor Trp binding activates Repressor Final State: OFF Final State: OFF

tryptophan

Features of the 5’ UTRFeatures of the 5’ UTR

•Contains complementary sequences that can form Contains complementary sequences that can form hairpin structures when transcribed into RNA hairpin structures when transcribed into RNA

•Codes for a stretch of U nucleotides that can act Codes for a stretch of U nucleotides that can act asas a termination signal after a hairpin structure a termination signal after a hairpin structure

•Codes for several Trp codons as part of an Codes for several Trp codons as part of an unstable protein product unstable protein product

Alternative RNA Structures from 5’ UTRAlternative RNA Structures from 5’ UTR

Termination signal due to Termination signal due to hairpin formed by 3+4 pairing hairpin formed by 3+4 pairing followed by string of uracils followed by string of uracils

No terminationNo terminationsignal formedsignal formed

Formation of termination signal depends on Formation of termination signal depends on level of tryptophan carried by tRNA in the cell. level of tryptophan carried by tRNA in the cell.

AttenuationAttenuationPremature Termination of TranscriptionPremature Termination of Transcription

Ribosome translates trp codons, preventing 2+3 pairing

3+4 pairing forms terminator

AntiterminationAntitermination

Ribosome stalls at trp codons,allowing 2+3 pairing

Transcription continuestoward trp E, D, C. B, A

Summary of Trp Operon RegulationSummary of Trp Operon Regulation

Level of Level of TryptophanTryptophan

Trp OperonTrp Operon

LowLow

HighHigh

OnOnTrp repressor inactive Trp repressor inactive Lack of attenuation leads to high rate of Lack of attenuation leads to high rate of mRNA productionmRNA production

OffOffTryptophan + repressor = Active repressor Tryptophan + repressor = Active repressor Reduction of mRNA production by attenuationReduction of mRNA production by attenuation