THE PROBLEMTHE PROBLEM

Prokaryotes must accomplish Prokaryotes must accomplish specialized functions in one specialized functions in one unspecialized cellunspecialized cell

OptionsOptions Have all gene products functioning at all Have all gene products functioning at all

times (times (constitutiveconstitutive expression) expression) Turn on genes only as they are needed Turn on genes only as they are needed

((inducibleinducible expression) expression) Are examples of both types of Are examples of both types of

expressionexpression

Control of Gene Function Control of Gene Function Control mRNA expression and lifetimeControl mRNA expression and lifetime

Deviations from consensus promoter sequencesDeviations from consensus promoter sequences Activator proteinsActivator proteins UP elementsUP elements

REMEMBER: prokaryotic mRNAs are REMEMBER: prokaryotic mRNAs are polycictronic, polycictronic, can have several genes can have several genes involved in a metabolic pathway expressed involved in a metabolic pathway expressed together (coordinated expression) together (coordinated expression)

Control translation and degradation of Control translation and degradation of protein productprotein product Half-life of protein Half-life of protein Position of cistron in polycistronic mRNAPosition of cistron in polycistronic mRNA Shine-Dalgarno deviationsShine-Dalgarno deviations

Regulation (cont’d)Regulation (cont’d) Negative regulation—Protein Negative regulation—Protein

((repressorrepressor) inhibits transcription (Ex. ) inhibits transcription (Ex. LexA).LexA). InducerInducer– binds to repressor, alters form, – binds to repressor, alters form,

reduces affinity for target, allows reduces affinity for target, allows expression of gene.expression of gene.

Sometimes, small molecule required for Sometimes, small molecule required for repressor activity. repressor activity.

Positive regulation—Positive regulation—ActivatorActivator proteinincreases transcription rate. proteinincreases transcription rate. Generally bound to a smaller signal Generally bound to a smaller signal molecule. molecule.

Regulation of Enzyme Regulation of Enzyme ActivityActivity

Degradation of Degradation of enzymeenzyme

Feedback Feedback inhibition– inhibition– generally a form generally a form of allosteric of allosteric inhibitioninhibition

Remember: the Remember: the cell is web of cell is web of competing competing pathways. pathways.

TheThe lac lac Operon Operon

Lactose—A disccharide hydrolyzed to Lactose—A disccharide hydrolyzed to glucose and galactose .glucose and galactose .

Lactose metabolizing enzymes Lactose metabolizing enzymes expresse as a polycistronic message expresse as a polycistronic message ( ( lacZ, lacY, lacAlacZ, lacY, lacA).).

Is an inducible Is an inducible operonoperon.. Consists of Consists of

Regulatory components Regulatory components Structural componentsStructural components

The PlayersThe Players RegulatoryRegulatory

Promoter (P)Promoter (P) Operator (O)Operator (O) LacILacI

StructuralStructural lacZlacZ lacYlacY lacAlacA

In The Absence of LactoseIn The Absence of Lactose

Repressor tetramer binds operator, prevents transcription

No reason for expression

is repressed

In The Presence of Lactose In The Presence of Lactose

Conformational change caused by inducer reduces affinity of repressor/inducer for operator

Role of CRPRole of CRP·cAMP·cAMP Expression of Expression of laclac operon operon

(+) Glucose (-) Lactose= No expression(+) Glucose (-) Lactose= No expression (+) Glucose (+) Lactose= Low to no expression(+) Glucose (+) Lactose= Low to no expression (-) Glucose (+) Lactose= High expression(-) Glucose (+) Lactose= High expression

When [glucose] is high, [cAMP] is low and When [glucose] is high, [cAMP] is low and vice vice versa.versa.

CCyclic AMP yclic AMP RReceptor eceptor PProtein forms a complex rotein forms a complex with cAMP and binds at a site near the promoter.with cAMP and binds at a site near the promoter. Strongly increases expressionStrongly increases expression Mechanism: causes bending of DNA, allows RNA pol 2 Mechanism: causes bending of DNA, allows RNA pol 2

points of caontactpoints of caontact

CAPCAP·cAMP Mechanism·cAMP Mechanism CAP-sensitive promoters usually weakCAP-sensitive promoters usually weak CAPCAP·cAMP Bends DNA, allowing RNA pol to ·cAMP Bends DNA, allowing RNA pol to

bind at two points, bind at two points, stabilizing interactionstabilizing interaction May also interact with C-terminal domain May also interact with C-terminal domain

of sigmaof sigma

LEGEND:

Purple- CAP·cAMP

Red- RNA pol

Blue- Sigma

Galactose OperonGalactose Operon Regulates catabolism of galactoseRegulates catabolism of galactose

3 cistrons encoding structural proteins3 cistrons encoding structural proteins 2 promoters (P1 and P2)2 promoters (P1 and P2) 2 operators2 operators Repressor (Repressor (gal Rgal R))

Gal Operon Regulation Gal Operon Regulation Effect of cAMP levelsEffect of cAMP levels

CAPCAP·cAMP regulates transcription from two ·cAMP regulates transcription from two promoters in opposite wayspromoters in opposite ways

CAPCAP·cAMP activates from P1, inhibits from P2 ·cAMP activates from P1, inhibits from P2 when when [cAMP][cAMP] transcribe from P1, when [cAMP] transcribe from P1, when [cAMP] transcribes form P2.transcribes form P2.

As long as no repression, level of As long as no repression, level of GalGal mRNA constant mRNA constant

RegulationRegulation Repressor- product of Repressor- product of galgal R R Inhibits from both operators Inhibits from both operators Galactose acts as inducerGalactose acts as inducer If galactose absent, both promoters inactiveIf galactose absent, both promoters inactive

Gal OperonGal Operon

One unit of the One unit of the galR dimer binds galR dimer binds to each operatorto each operator

Induces Induces conformational conformational change, prevents change, prevents transcriptiontranscription

Possible structuresNote: dimer responsible for repression

Ara OperonAra Operon Dual action Dual action

regulatory protein- regulatory protein- AraC AraC (-) arabinose(-) arabinose

RepressesRepresses (+) arabinose(+) arabinose

ActivatesActivates AraI AraI

AraIAraI11

AraIAraI22

OperatorsOperators AraOAraO11- regulates - regulates

AraCAraC AraOAraO22- regulates - regulates

AraBADAraBAD

Two operators AraIAraIIn absence of arabinose- AraC dimer causes loop by joining I1 and O2. no transcription

With arabinose, shape change causes dimer to sit on I1 and I2, allowing transcription

Ara operon 2Ara operon 2 NOTE: CAPNOTE: CAP·cAMP binding site. Increases ·cAMP binding site. Increases

transcription. transcription. Autoregulation of Autoregulation of AraCAraC

AraC AraC transcribed from P transcribed from Pcc.. PPcc regulated by O regulated by O11. . As level of AraC rises, binds to AraOAs level of AraC rises, binds to AraO11 and and

prevents transcription from Pprevents transcription from Pcc. . prevents wasteful accumulation of prevents wasteful accumulation of

repressorrepressor Is an example of autoregulationIs an example of autoregulation

Are other models Are other models

TrpTrp Operon Operon Encodes enzymes necessary for Trp Encodes enzymes necessary for Trp

synthesissynthesis encodes a set of encodes a set of anabolicanabolic enzymes rather than enzymes rather than

catabolic enzymes.catabolic enzymes. Anabolic enzymes are generally turned off by Anabolic enzymes are generally turned off by

presence of a product (feedback inhibition)presence of a product (feedback inhibition) In addition to repression, system shows In addition to repression, system shows

attenuationattenuation, a finer level of control. , a finer level of control. StructureStructure

5 structural genes5 structural genes3 enzymes3 enzymes Promoter and operator precede structural genesPromoter and operator precede structural genes In absence of Trp, In absence of Trp, TrpRTrpR protein is inactive protein is inactive

Tryptophan Operon Tryptophan Operon RepressionRepression

Negative control of operon:Negative control of operon: Low tryptophanLow tryptophan No repressionNo repression transcriptiontranscription

Positive control of operon:Positive control of operon: High tryptophanHigh tryptophan Tryptophan (a Tryptophan (a corepressorcorepressor) combines ) combines

with free repressor dimer (with free repressor dimer (aporepressor aporepressor dimerdimer)=repressor dimer)=repressor dimer

transcription blockedtranscription blocked

Attenuation: A Finer Level of Attenuation: A Finer Level of ControlControl

Trp operon expression also regulated Trp operon expression also regulated by by attenuation,attenuation, a much finer level of a much finer level of control.control.

Trp operon featuresTrp operon features Repression very weakRepression very weak

transcription could occur even in presence transcription could occur even in presence of repressorof repressor

Very energy expensiveVery energy expensive Attenuation increases expression 10-foldAttenuation increases expression 10-fold Result: Trp operon expression spans a Result: Trp operon expression spans a

700-fold range (from inactive to fully 700-fold range (from inactive to fully active)active)

Attenuation MechanismAttenuation Mechanism Special sequences prior between promoter Special sequences prior between promoter

and structural geneand structural gene Trp leaderTrp leader

Has translation start siteHas translation start site 2 Trp codons in a row (very rare)2 Trp codons in a row (very rare)

Trp attenuatorTrp attenuator Has transcription termination sequenceHas transcription termination sequence

These sequences weaken (attenuate) These sequences weaken (attenuate) transcription when trp is abundanttranscription when trp is abundant

Operates by causing premature Operates by causing premature termination of transcriptiontermination of transcription

REMEMBER: transcription and translation REMEMBER: transcription and translation occur simultaneously in prokaryotes occur simultaneously in prokaryotes

Attenuation Mechanism 2Attenuation Mechanism 2 Different hairpin configurationsDifferent hairpin configurations

Configuration Configuration 11—Two hairpins, 4 stems—Two hairpins, 4 stems Configuration Configuration 22- One hairpin, two stems- One hairpin, two stems

Configuration 1 is more stableConfiguration 1 is more stable Translation begins as soon as Translation begins as soon as TrpTrp

leader transcript emergesleader transcript emerges If Trp is in short supplyIf Trp is in short supply Ribosome will stall over Stem 1Ribosome will stall over Stem 1

If Trp AbundantIf Trp Abundant

Ribosome Ribosome translates, hits translates, hits termination codon, termination codon, falls offfalls off

Allows formation of Allows formation of 2 hairpins2 hairpins

One contains One contains intrinsic terminator intrinsic terminator

RNA pol falls offRNA pol falls off

If Trp is Scarce- Overriding If Trp is Scarce- Overriding AttenuationAttenuation

Ribosome will stall Ribosome will stall over Trp codons in over Trp codons in Trp leader sequenceTrp leader sequence

Double hairpin Double hairpin can’t form, only can’t form, only single hairpin single hairpin configurationconfiguration

Allows RNA pol to Allows RNA pol to transcribe through transcribe through termination termination sequencessequences

The OperonsThe Operons

OPEROOPERONN

INDUCEINDUCERR

RepressRepressor geneor gene

CAPCAP COMMENTSCOMMENTS

laclac lactoselactose lacIlacI ++ One promoter, one One promoter, one operatoroperator

galgal galactosgalactosee

galRgalR ++ Two operators, 2 Two operators, 2 promoterspromoters

araara arabinosarabinosee

araCaraC ++ araC protein acts as araC protein acts as repressor and activatorrepressor and activator

TrpTrp Low TrpLow Trp trpR trpR ((aporepressoaporepressorr))

-- Anabolic, Trp is Anabolic, Trp is corepressorcorepressor, , attenuationattenuation