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    BY:PRIYANKA PATEL

    Msc.(B.T) SEM-I

    ROLL NO:47

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    Cells must onlyexpress genes when needed Geneexpression (transcription, translation)

    takes uplargeamounts of cellularenergyandresources

    Cells live frugallifestyles they conserveenergyandresources

    So genes will only beexpressed when theirproducts are needed.

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    Bacteria control genes at the transcriptional

    level

    In other words, the gene is either transcribed or

    not, based on certain external stimuli

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    Francois Jacob & Jacques Monod

    first to describe operon system

    coined thephrase operon

    2005-2006

    1961 /1965

    Francois JacobJacques Monod

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    An operon is a collection ofprokaryotic genes

    transcribed together on a single mRNAtranscript to servea singlepurpose

    Composed of

    An operator, an on-off switch

    A promoter

    Genes for metabolic enzymes

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    A Region of DNA that interects with a

    repressorprotein to control theexpression of

    a gene ora group of gene Found within thepromoterregion or

    between thepromoterand geneencodingregions.

    On-off switch to gene

    Controls theaccess of RNA polymerase +

    Gene

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    A DNA sequenceat which RNA polymeres

    may bind ,leading to initiation of

    transcription Near the operator

    Onepromoter controls transcription ofallgene in operon

    Transcribedas 1 unit & a single mRNA ismade

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    It code for theenzymes themselves. The structural genes ofan operon usuallylies

    adjacent to oneanother,and the RNA

    polymarase moves from one structural gene tothe next , transcribing all of the genes into asingal mRNA.

    This extended mRNA is then translated into the

    various individualenzymes of the metabolicpathway. Turning on one gene turns on all theproducing

    gene ofan operon.

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    Catabolic operon:An operon composed of

    genes whoseproducts degrade organic

    compounds.

    Biosynthetic operon:An operon composed ofgenes whoseproducts are involved in

    synthesizing compounds,such as amino acidsor vitamins,rather than degrading them.

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    Inducer: A signal molecule that, when bind to

    aregulatoryprotein, produces an increase in

    theexpression ofa given gene

    Repressor: Theprotein that binds to theregulatory sequence or operator fora

    gene,blocking its transcription

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    Corepressor:The small molecule that

    associate with an aporepressor to form an

    activerepressor.(e.g.,tryptophan is thecorepressor of the trp operon).

    Aporepressor:A repressor in an inactive form

    ,without its corepressor.

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    A repressible operon is one that is usually on;

    binding ofarepressor shuts off transcription

    Thetrp operon is arepressible operon An inducible operon is one that is usually off;

    a molecule calledan inducer inactivates therepressorand turns on transcription

    The classic example ofan inducible operon isthelac operon

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    Inducibleenzymes usually function in catabolic

    pathways; their synthesis is induced bya

    chemical signal

    Repressibleenzymes usually function inanabolic pathways; their synthesis is repressed

    by high levels of theendproduct

    Regulation of thetrp andlac operons involvesnegative control of genes because operons areswitched off by theactive form of therepressor

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    Some operons arealso subject to positivecontrol through a stimulatoryprotein, such as

    cataboliteactivatorprotein (CAP), an activator

    of transcription When glucose (apreferred food source ofE.

    coli) is scarce,CAP is activated by binding with

    cyclic AMP ActivatedCAP attaches to thepromoter of the

    lacoperon and increases theaffinity of RNA

    polymerase, thus accelerating transcription

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    When glucoselevels increase,CAP detachesfrom thelac operon, and transcription returns

    to a normalrate

    CAP helps regulate other operons that encodeenzymes used in catabolic pathways

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    Thetrp operon contains 5 structural genes

    ,TRP -E,D,C,A,B.

    These geneencode for 3 enzymes that turnchorismic acid into Tryptophan.

    The operon is not expressedwhen the cellcontains sufficient amounts of tryptophan.

    The operon is expressed when levels oftryptophan arelow.

    18

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    Thetrp operon is negativelyregulated by thetrp repressor protein

    trp repressor binds to the operator toblock transcription

    binding ofrepressorto the operatorrequiresacorepressorwhich istryptophan

    low levels of tryptophan prevent therepressor from binding to the operator

    19

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    DNA

    Tryptophan (co-

    repressor)

    TrpR protein

    subunits

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    Converts chorismate to anthranilate usingammonia.

    Anthranilate is an amino acid that is laterbroken down into another amino acid,Trysoine.

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    A type ofGlycosyltransferase thatparticipates in the synthesis of Tryptophan

    Produces an amino acid calledAnthranilatewhich ,by the help of TRP E, is broken downinto anotheramino acid, Trysoine.

    Converts -(5-phospho-D-ribosyl)-

    anthranilateanddiphosphate toanthranilateand 5-phospho-alpha-D-ribose1-diphosphate.

    It is 1 of the 2 components that together

    encodeAnthranilate synthetase

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    Involved in the synthesis of tryptophan and

    amino acids.

    It is a type oflyase that cleaves carbon-carbon bond.

    this abilityallows it to converts 1-(2-

    carboxyphenylamino)-1-deoxy-D-ribulose5-phosphate to C(1)-(3-indolyl)-glycerol 3-

    phosphateand carbon dioxideand water

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    Thereare two beta subunits which form a

    dimer

    Betadimer catalyzes the formation of L-tryptophan from L-serineand 1-(indol-3-

    yl)glycerol 3-phosphate

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    Tryptophan synthase is an enzyme found in

    plants and bacteria

    Thereare two separatealpha subunits They catalyze the formation of indoleand

    glyceraldehyde 3-phosphate fromindoleglycerolphosphate in tryptophan

    biosynthesis, which is needed by the Bsubunits.

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    Tryptophan present

    Regulator Gene Promoter Operator Structural GenesAttenuator

    trpRmRNA

    RNA Polymerase

    NOTRANSCRIPTION

    TrpR protein

    (homodimer)

    + tryptophan

    (corepressor)

    TrpR aporepressor

    + corepressor(can

    bind to operator)

    Q: Why might the cell want

    to produce an aporepressor

    that is only activated by the

    operons end product?

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    Tryptophan Absent

    Regulator Gene Promoter Operator Structural GenesAttenuator

    trpRmRNA

    RNA Polymerase

    TRANSCRIPTION

    TrpR protein

    (homodimer)TrpR aporepressor

    (cannot bind to

    operator)

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    Attenuator:A region of DNA upstream from

    one or more structural genes, wherepremature

    transcription termination(attenuation)can occur.

    The Attenuation Mechanism oftrp Operon

    Mechanism of Attenuation

    When Ribosomereaches Trp

    codon

    Trp is scarce

    Trp is abundant

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    Attenuation model in Trp starved cells.

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    Attenuation model in Trp non-starved cells

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    Generality of Attenuation

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    TheTrpR repressor negativelyregulates not

    only the transcription of the trp operon but

    also the transcription of its own gene,trpR. In theabsence of theTrpR protein,the

    transcription of the trpR gene is about 5times higher then in thepresence of TrpR.

    When theproduct ofa generegulates the

    expression of its own gene,it is called

    autoregulation.

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    L- ara operon was the first e.g. ofpositiveregulation in bacteria to bediscovered.Its

    usually called theara operon. Theara operon contains 3 structuralgene,ara-A,B,D together with theI andOsites.

    The gene of this operon areresponsible forconverting L- arabinose into D-xylulos-5-phosphate,which can be used by otherpathways.

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    The operon is not expressedwhen the

    arabinose is absent in the cell.

    The operon is expressed when thearabinoseis present in the cell.

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    P

    t

    Pt

    araC araB araA araD

    Activator/Repressor

    I1 I2

    AraCdimer

    CRP

    O2 O1

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    araO1:Itis an operator site. AraC binds to this siteandrepresses its own transcription from thePCpromoter. In thepresence ofarabinose, however,AraC boundat this site helps to activateexpression

    of thePBAD promoter. araO2:Itis also an operator site. AraC boundat this

    site can simultaneously bind to thearaIsite torepress transcription from thePBAD promoter

    araI:Itis also the inducer site. AraC boundat thissite can simultaneously bind to thearaO2 site torepress transcription from thePBAD promoter. Inthepresence ofarabinose, however, AraC boundat

    this site helps to activateexpression of thePBADromoter.

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    CRP binds to theCRP binding site. It does not

    directlyassist RNA polymerase to bind to the

    promoter in this case. Instead, in thepresence ofarabinose, it promotes the

    rearrangement ofAraC when arabinose ispresent from a state in which it represses

    transcription of thePBAD promoter to one inwhich it activates transcription of thePBAD

    promoter.

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    ara A :Encode L- Arabinose isomerase whichconverts L- Arabinose into L- Ribulose.

    ara B :Encode L- Ribulosekinase whichphosphorylates L- Ribulose to L-Ribulose-5-

    phosphate.

    ara D :Encode L-Ribulose-5-phoshate

    epimerase which converts L-Ribolose-5-phosphate into D-Xylulose-5-phosphate.which can then be metabolized via the

    pentosephosphatepathway.

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    When arabinose is absent, there is no need to

    express the structural genes. AraC does thisby binding simultaneously to araIandaraO2.

    As aresult the intervening DNA is looped.

    These two events blockaccess to thePBADpromoter which is, in any case, a very weak

    promoter (unlike thelacpromoter):

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    Arabinose absentNegative control

    Blocked

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    When arabinose is present, it binds to AraC

    andallosterically induces it to bind to araIinsteadaraO2. Ifglucose is also absent, then

    thepresence ofCRP bound to its site

    between araO1 andaraIhelps to break theDNA loopandalso helps AraC to bind to araI:

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    Arabinose present Positive control

    araB A D

    O1 CRP araI

    araC

    PC PBAD

    O2

    Transcription

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    AraC can bind to three sites (araO, araI1, andaraI2) with different affinities

    46

    Fig. 15.23

    (a) No arabinose present:

    When AraC is bound to araO and toaraI1, looping of DNA occurs and

    prevents transcription

    (b) Arabinose present:Arabinose causes allosteric change in

    AraC so that it cannot bind to araO

    AraC interacts with RNA polymerase

    only when both araI1 and araI2are

    occupied

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    TheAraCprotein not onlyregulate theara operon

    but also negativelyautoregulates its own

    transcription. If the concentration of AraC becomes too high,its

    synthesis willagain berepressed.

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    Thereare several mutations that couldpreventtheexpression of thearabinose operon genesandeven cause the cell to die

    . A mutation in the araA genewill cause thebacterial cell to becomearabinose negative. Thismeans that the bacterium can no longer usearabinoseas a carbon source. It will not utilize itif the cell is grown in a minimal media containingarabinose,.

    A mutation in the ara B genewillalso result inthe same state. However, amutation in the araD genewillresult in cell death.

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    After the ara B geneproduct degrades L-ribulose to L-ribulose-5-phosphate, epimerase isnot synthesizedand cannot breakdown L-

    ribulose-5-phosphate which accumulates in thecell. L-ribulose-5-phosphateis toxic to the cell when

    present in high levels. Mutations couldalso occurin the araCgene

    causing thepromoters, PBAD and Pc, to becomeinactiveand thearabinose operon remainspermanently repressed.

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    It is also regulated through cataboliterepression , sothe gene forarabinose utilization are not expressed ifthe medium contains a better carbon source.

    CAP may help open theloop of DNA created whenAraC bind to araO2 andara1.opening theloop mayprevent AraC from binding to araO2 andaraI1,facilating the binding of AraC to araI1 andaraI2 andtheactivation of transciption from pBAD.

    Thus, the nt of glucose or another carbon sourcebetter then arabinose enhances the transcription ofthe ara operon.

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    Scientists modified thearabinose operon in

    pGLO to express theGFP gene

    TagCells (to detect specific cells) Act as areporter gene

    Source ofbioluminescence whenexposed to

    UV light

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    Molecular Biology of theGene:Watson et al.

    MolecularGenetics of Bacteria: Synder &

    Champness www.google.in

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