Molecular biology - Chapter 7: Operons: Fine control of bacterial transcription

Molecular BiologyFourth EditionChapter 7Operons: Fine Control ofBacterial TranscriptionLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.17.1 The lac OperonThe lac operon was the first operon discoveredContains 3 genes coding for E. coli proteins that permit the bacteria to use the sugar lactoseGalactoside permease which transports lactose into the cellsb-galactosidase cuts the lactose into galactose and glucoseGalactoside transacetylase whose function is unclear2Genes of the lac OperonGenes are grouped:lacZ = b-galactosidaselacY = galactoside permeaselacA = galactoside transacetylaseAll 3 genes are transcribed together producing 1 mRNA, a polycistronic message that starts from a single promoterEach cistron, or gene, has its own ribosome binding siteEach cistron can be transcribed by separate ribosomes that bind independently of each other3Control of the lac OperonThe lac operon is tightly controlled, using 2 types of controlNegative control, like the brake of a car, must remove the repressor from the operatorAn activator, additional positive factor, responds to low glucose by stimulating transcription of the lac operon4Negative Control of the lac OperonNegative control indicates that the operon is turned on unless something turns it off and stops itThe off-regulation is done by the lac repressor Product of the lacI geneTetramer of 4 identical polypeptidesBinds the operator just right of promoter5lac RepressorWhen repressor binds the operator, operon is repressedOperator and promoter are contiguousRepressor bound to operator prevents RNA polymerase from binding to the promoterAs long as no lactose is available, lac operon is repressed6Negative Control of the lac Operon7Inducer of the lac OperonThe repressor is an allosteric protein Binding of one molecule to the protein changes shape of a remote site on that proteinAltering its interaction with a second moleculeInducer (one molecule) of lac operon binds the repressorCausing the repressor to change conformation that favors release from the operator (the second molecule)The inducer is allolactose, an alternative form of lactose8Inducer of the lac OperonInducer (one molecule) of lac operon binds the repressorThe inducer is allolactose, an alternative form of lactose9Discovery of the OperonDuring the 1940s and 1950s, Jacob and Monod studied the metabolism of lactose by E. coliThree enzyme activities / three genes were induced together by galactosidesConstitutive mutants need no induction, genes are active all the timeMerodiploids are partial diploid bacteria10Effects of Regulatory Mutations:Wild-type and Mutated Repressor11Effects of Regulatory Mutations:Wild-type and Mutated Operator with Defective Binding12Repressor-Operator InteractionsUsing a filter-binding assay, the lac repressor binds to lac operatorA genetically defined constitutive lac operator has lower than normal affinity for the lac repressorSites defined by two methods as the operator are in fact the same13The Mechanism of RepressionThe repressor does not block access by RNA polymerase to the lac promoterPolymerase and repressor can bind together to the lac promoterPolymerase-promoter complex is in equilibrium with free polymerase and promoter14lac Repressor and Dissociation of RNA Polymerase From PromoterWithout competitor, dissociated polymerase returns to promoterHeparin and repressor prevent reassociation of polymerase and promoterRepressor prevents reassociation by binding to the operator adjacent to the promoterThis blocks access to the promoter by RNA polymerase15Effects of Regulatory Mutations:Wild-type and Mutated Operon Binding Irreversibly16Mechanism SummaryTwo hypotheses of mechanism for repression of the lac operonRNA polymerase can bind to lac promoter in presence of repressorRepressor will inhibit transition from abortive transcription to processive transcriptionRepressor, by binding to operator, blocks access by the polymerase to adjacent promoter17lac OperatorsThere are three lac operatorsThe major lac operator lies adjacent to promoterTwo auxiliary lac operators - one upstream and the other downstreamAll three operators are required for optimum repression The major operator produces only a modest amount of repression18Catabolite Repression of the lac OperonWhen glucose is present, lac operon is in a relatively inactive state Selection in favor of glucose attributed to role of a breakdown product, cataboliteProcess known as catabolite repression uses a breakdown product to repression the operon 19Positive Control of lac OperonPositive control of lac operon by a substance sensing lack of glucose that responds by activating lac promoterThe concentration of nucleotide, cyclic-AMP, rises as the concentration of glucose drops20Catabolite Activator ProteincAMP added to E. coli can overcome catabolite repression of lac operonAddition of cAMP lead to activation of the lac gene even in the presence of glucosePositive controller of lac operon has 2 parts:cAMPProtein factor is known as:Catabolite activator protein or CAPCyclic-AMP receptor protein or CRPGene encoding this protein is crp21Stimulation of lac OperonCAP-cAMP complex positively controls the activity of b-galactosidaseCAP binds cAMP tightlyMutant CAP not bind cAMP tightly preparedCompare activity and production of b-galactosidase using both complexesLow activity with mutant CAP-cAMP 22The Mechanism of CAP ActionCAP-cAMP complex binds to the lac promoterMutants whose lac gene is not stimulated by complex had the mutation in the lac promoterMapping the DNA has shown that the activator-binding site lies just upstream of the promoterBinding of CAP and cAMP to the activator site helps RNA polymerase form an open promoter complex23CAP Plus cAMP ActionThe open promoter complex does not form even is RNA polymerase has bound the DNA unless the CAP-cAMP complex is also bound24CAP Binding sites for CAP in lac, gal and ara operons all contain the sequence TGTGASequence conservation suggests an important role in CAP bindingBinding of CAP-cAMP complex to DNA is tightCAP-cAMP activated operons have very weak promotersTheir -35 boxes are quite unlike the consensus sequenceIf these promoters were strong they could be activated even when glucose is present25RecruitmentCAP-cAMP recruits polymerase to the promoter in two stepsFormation of the closed promoter complexConversion of the closed promoter complex into the open promoter complexCAP-cAMP bends its target DNA by about 100° when it binds26CAP-cAMP-Promoter Complexes27Proposed CAP-cAMP Activation of lac TranscriptionThe CAP-cAMP dimer binds to its target site on the DNAThe aCTD (a-carboxy terminal domain) of polymerase interacts with a specific site on CAPBinding is strengthened between promoter and polymerase287.2 The ara OperonThe ara operon of E. coli codes for enzymes required to metabolize the sugar arabinoseIt is another catabolite-repressible operon29Features of the ara OperonTwo ara operators exist:araO1 regulates transcription of a control gene called araCaraO2 is located far upstream of the promoter is controlsCAP-binding site is 200 bp upstream of the ara promoter, yet CAP stimulates transcriptionThis operon has another system of negative regulation mediated by the AraC protein30The ara Operon Repression LoopThe araO2 operator controls transcription from a promoter 250 downstreamDoes the DNA between the operator and the promoter loop out?31The ara Control Protein The AraC, ara control protein, acts as both a positive and negative regulatorThere are 3 binding sitesFar upstream site, araO2araO1 located between -106 and -144araI is really 2 half-sitesaraI1 between -56 and -78 araI2 -35 to -51 Each half-site can bind one monomer of AraC32The araCBAD OperonThe ara operon is also called the araCBAD operon for its 4 genesThree genes, araB, A, and D, encode the arabinose metabolizing enzymesThese are transcribed rightward from the promoter araPBADOther gene, araCEncodes the control protein AraCTranscribed leftward from the araPc promoter 33AraC Control of the ara OperonIn absence of arabinose, no araBAD products needed, AraC exerts negative controlBinds to araO2 and araI1Loops out the DNA in betweenRepresses the operonPresence of arabinose, AraC changes conformationIt can no longer bind to araO2Occupies araI1 and araI2 insteadRepression loop brokenOperon is derepressed34Control of the ara Operon35Positive Control of the ara OperonPositive control is also mediated by CAP and cAMPThe CAP-cAMP complex attaches to its binding site upstream of the araBAD promoterDNA looping would allow CAP to contact the polymerase and thereby stimulate its binding to the promoter36ara Operon SummaryThe ara operon is controlled by the AraC proteinRepresses by looping out the DNA between 2 sites, araO2 and araI1 that are 210 bp apartArabinose can derepress the operon causing AraC to loosen its attachment to araO2 and bind to araI2Break the loop and allow transcription of operonCAP and cAMP stimulate transcription by binding to a site upstream of araIAraC controls its own synthesis by binding to araO1 and prevents leftward transcription of the araC gene377.3 The trp OperonThe E. coli trp operon contains the genes for the enzymes the bacterium needs to make the amino acid tryptophanThe trp operon codes for anabolic enzymes, those that build up a substanceAnabolic enzymes are typically turned off by a high level of the substance producedThis operon is subject to negative control by a repressor when tryptophan levels are elevatedtrp operon also exhibits attenuation38Tryptophan’s Role in Negative Control of the trp OperonFive genes code for the polypeptides in the enzymes of tryptophan synthesisThe trp operator lies wholly within the trp promoterHigh tryptophan concentration is the signal to turn off the operonPresence of tryptophan helps the trp repressor bind to its operator39Negative Control of the trp OperonWithout tryptophan no trp repressor exists, just the inactive protein, aporepressorIf aporepressor binds tryptophan, changes conformation with high affinity for trp operatorCombine aporepressor and tryptophan to have the trp repressorTryptophan is a corepressor40Attenuation in the trp Operon 41Mechanism of AttenuationAttenuation imposes an extra level of control on an operon, more than just the repressor-operator systemOperates by causing premature termination of the operon’s transcript when product is abundant42Defeating AttenuationAttenuation operates in the E. coli trp operon as long as tryptophan is plentifulIf amino acid supply low, ribosomes stall at the tandem tryptophan codons in the trp leadertrp leader being synthesized as stalling occurs, stalled ribosome will influence the way RNA foldsPrevents formation of a hairpinThis is part of the transcription termination signal which causes attenuation43Overriding Attenuation447.4 RiboswitchesSmall molecules can act directly on the 5’-UTRs of mRNAs to control their expressionRegions of 5’-UTRs capable of altering their structures to control gene expression in response to ligand binding are called riboswitches45Riboswitch ActionRegion that binds to the ligand is an aptamerAn expression platform is another module in the riboswitch which can be:Terminator Ribosome-binding siteAnother RNA element that affects gene expressionOperates by depressing gene expressionSome work at the transcriptional levelOthers can function at the translational level46Model of Riboswitch ActionFMN binds to aptamer in a riboswitch called the RFN element in 5’-UTR of the ribD mRNABinding FMN, base pairing in riboswitch changes to create a terminatorTranscription is attenuatedSaves cell energy as FMN is a product of the ribD operon47