Presentation on theme: "Operons: Fine Control of Bacterial Transcription"— Presentation transcript:
1 Operons: Fine Control of Bacterial Transcription Chapter 7
2 Why is Operon system important? 3000 genes in E.coli – some products are in great demandSome genes are turned off – rarely neededCannot leave all genes on all the time – takes energy for transcription and translation – drain cell of energyScientists – use Operon system
3 The lac Operon The lac operon - first operon discovered Contains 3 genes coding for E. coli proteins that permit the bacteria to use the sugar lactoseGalactoside permease (lacZ) - transports lactose into the cellsb-galactosidase (lacY) cuts the lactose into galactose and glucoseGalactoside transacetylase (lacA)- function is unclearIn a flask containing glucose and lactose, the cells exhaust glucose and stop growing. For a short time it appears that it cannot adjust to lactose, but resumes growth after some time and turn on their lac operon to begin to accumulate their enzymes needed to metabolize lactose.Lactose is composed of two simple sugars galactose and glucose joined by beta galactosidic bond.
4 lac OperonAll 3 genes are transcribed together producing 1 mRNA - a polycistronic message that starts from a single promoterCistron or gene - has its own ribosome binding siteA polycistronic message with information from more than one gene. Each cistron can be translated by separate ribosomes that bind independently of each other
5 Control of the lac Operon The lac operon is tightly controlled - using 2 types of controlNegative control - like the brake of a car - binds the repressor to the operatorPositive control – like accelerator pedal – removes repressor from operatorActivator - additional positive factor - responds to low glucose by stimulating transcription of the lac operonThe brake in negative control is a protein called lac repressor which keeps operon turned off or repressed as long as lactose is absent. Because it would be wasteful for the cell to produce enzymes needed to use an absent sugar. An additional factor activator is needed to activate operon. It responds to low glucose levels by stimulating transcription of lac operon, but high glucose levels keeps concentration of activator low so transcription of operon cannot be stimulated. The advantage of this positive control is that it keeps operon turned off when level of glucose is high as bacteria metabolize glucose more easily than lactose and it will be wasteful for them to activate the lac operon in presence of glucose
7 Negative Control of the lac Operon Negative control - operon is turned on unless something turns it off and stops itThe off-regulation is done by the lac repressorProduct of the lacI geneTetramer of 4 identical polypeptidesBinds the operator just right of promoterRepressor is the protein that turns operon off. When repressor binds the operator, operon is repressed. The operator and promoter are contiguous. When repressor bind to operator then it prevents RNA polymerase from binding to the promoter and transcribing the operon.As long as no lactose is available, lac operon is repressed
8 lac repressor When repressor binds the operator - operon is repressed Operator and promoter are contiguousRepressor bound to operator prevents RNA polymerase from binding to the promoterAs long as no lactose is available - lac operon is repressed
9 Inducer of lac operon The repressor is an allosteric protein Binding of one molecule (inducer – allolactose) to the protein (repressor) changes shape of a remote site on that proteinAltering its interaction with a second molecule (operator)Inducer (one molecule) of lac operon binds the repressor causing the repressor to change conformation that favors release from the operator (the second molecule). The inducer is allolactose, an alternative form of lactose. This happens in absence of lactose
10 Mechanism 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 promoter
11 Three lac operators Three lac operators The major lac operator lies adjacent to promoterTwo auxiliary lac operators - one upstream and the other downstreamAll three operators are required for optimum repressionThe major operator produces only a modest amount of repression
12 Role in repression of transcription by the three operators
13 Catabolite repression When glucose is present - lac operon is in an inactive stateSelection in favor of glucose attributed to role of a breakdown product – CataboliteCatabolite repression uses catabolite to repress the operonSelection in favor of glucose attributed to role of a breakdown product – Catabolite. The breakdown product is related to glucose or catabolite of glucose. This happens when both glucose and lactose are present. The bacteria will not use lactose until it metabolizes glucose
14 Positive Control of the lac Operon Positive control of lac operon by a substance sensing lack of glucose -activate lac promoterThe conc. of nucleotide - cyclic-AMP - rises as the concentration of glucose drops
15 Catabolite Activator Protein cAMP added to E. coli can overcome catabolite repression of lac operonPositive controller of lac operon has 2 parts:cAMP + protein factorProtein factor is known as:Catabolite activator protein or CAPCyclic-AMP receptor protein or CRPGene encoding this protein is crpAddition of cAMP led to activation of the lac gene even in the presence of glucose
16 The Mechanism of CAP Action CAP-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 complex
17 CAP-cAMP complexThe open promoter complex does not form even if RNA polymerase has bound to the DNA - till the CAP-cAMP complex is also boundCAP-cAMP recruits polymerase to the promoter in two stepsFormation of the closed promoter complexConversion of the closed promoter complex into the open promoter complex
18 CAPBinding sites for CAP in lac, gal and ara operons -contain the sequence TGTGABinding of CAP-cAMP complex to DNA is tightCAP-cAMP activated operons have very weak promotersTheir -35 boxes are quite unlike the consensus sequenceSequence conservation suggests an important role in CAP binding
19 CAP-cAMP Activation of lac Transcription The 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 polymerase
20 ara OperonThe 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 -78araI to -51
21 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
23 AraC Control of the ara Operon In absence of arabinose - no araBAD products - 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 derepressed
24 Positive Control of the ara Operon Positive control is also mediated by CAP and cAMPThe CAP-cAMP complex attaches to its binding site upstream of the araBAD promoter
25 The trp OperonThe E. coli trp operon contains the genes for the enzymes - to make the amino acid tryptophanThe trp operon codes for anabolic enzymesAnabolic enzymes are typically turned off by a high level of the substance producedOperon - negative control by a repressor - tryptophan levels are elevatedtrp operon also exhibits attenuationanabolic enzymes those that build up a substance. This operon is subject to negative control by a repressor when tryptophan levels are elevated
26 Tryptophan’s Role in Negative Control of the trp Operon Five genes code for the polypeptides in the enzymes of tryptophan synthesis – Chorismic acidThe trp operator lies wholly within the trp promoterPresence of high tryptophan helps the trp repressor bind to its operatorIn lac operon, the promoter and operator precede the genes and same is true for trp operon. In trp operon the operator lies within the promoter. In positive control of lac operon, the cell senses the presence of lactose by appearance of tiny amounts of its rearranged product – allolactose. This causes the repressor to fall off lac operator and derepress the operon.
27 Negative control of trp operon Without tryptophan no trp repressor exists -inactive protein - aporepressorIf aporepressor + tryptophan - changes conformation with high affinity for trp operatorCombine aporepressor and tryptophan to have the trp repressorTryptophan is a corepressor
28 Mechanism of Attenuation Attenuation 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 abundantThe reason for premature termination is attenuator contains a transcription stop signal (terminator): an inverted repeat followed by a string of eightA-T pairs in a row. Because of inverted repeat the transcript of this region would tend to engage in intramolecular base pairing forming hairpin.
29 Defeating Attenuation Attenuation 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 hairpin
31 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 riboswitchesRegion that binds to the ligand is an aptamer
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