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GeneExpression(operons) اھایحأ نمو

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GeneExpression(operons)

DONEBY:SaraAlzoubi&WessenZyoutLecturer:Dr.NabeelBasheer


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-Lasttimewetalkedaboutlacoperon(lactoseoperon),anyoperonconsistofstructuralgenesandregulatorygenes.

arefoundinthepromoter,incaseoflacregulatorysequenceThe-operonthereisalsoaregionwithinthepromotercalledoperatorandbeforethepromoterthereisaregioncalledLACI.

*lacIisasequencethatwillproducearepressorproteinsthatwillbindtotheoperator*

operonlacThearerelatedtoeachother,structuralgenesusuallythe-lacZ:,thesestructuralgenesarestructuralgenesconsistsofthree

lacA.lacY,and,

willproducethreeproteinswhicharerelatedtothethreegenesthese-composed of sugardisaccharideametabolismoflactose(

for sources of energyand both serve as and galactose glucoseyour cell )

cleaves(hydrolyses):itgalactosidase-βencodes*lacZ.galactoseandglucoseintolactosedisaccharidethe

transportlactoseinsidethe:itgalactosidepermease-BetaencodeslacYcell.*

:modifythelactosegalactosidetransacetylase-βsencodelacA*(acetylation)toproduceinducerfortheregulationofthisoperon.

consistofoperatorthatwillbindtoaregulatorysequencethe-t is produced from another sequence called proteins tharepressor

lac I(lac I found before the promoter).

RNAncetherepressorproteinbindstotheoperator,itwillprevento-POLYMERASEfrombindingandthusswitchingofftranscription.

,thecellrequirethisoperontobeswitched*Inthepresenceoflactose-Lactose(allo-on,verylittleamountoflactosewillbeconvertedtoAllo

repressorandpreventlactosewillfunctionasinducer,itwillbindtothethisrepressorfrombindingtotheoperatorthusRNA


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POLYMERASE(RNAPs)couldbindtothepromoterandtranscriptionwillbeswitchedon.

TranscriptionoftheoperonwillproducebigmRNAthatwillbe(enzymes)thatareneededtotranslatedintothethreeproteins

metabolizelactose.

theoperonisswitchingoff,WHY?,*intheabsenceoflactose

Becausethereisnoinducerandtherepressorproteinwillbeproduced.andbindtotheoperatorandblocksthebindingofRNAPs

ALLO-LACTOSE


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Anotherimportantitemintheoperon:

Allo-Lactose

Lactose

Polycistronic MessageinProkaryotes


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- CAP sitewillbindtoaproteincalledCataboliteactivatorprotein(CAP),thisbindingwillincreasetheRNAPsaffinitytothepromoterandincreasetranscription.

**CAPISNTALWAYESABLETOBINDDNA,IT'SREGULATEDBYTHELEVELOFGLUCOSE.

-CAMPisproducedwhenthereisnoenergy(glucose),soCAMPwillbehighanditwillbindtoCAP,thiscomplex(CAP-CAMPComplex)willbindtotheCAPSITEthusRNAPsbindingwillbehigh,RNAPsbindsitspromoteranditwilltranscribemRNA(consistofthreegenes)thatwillbetranslatedinto3differentproteins.

-whenglucoselevelarehigh,noCAMPismade,CAPcantbindtoDNAwithoutCAMP,sotranscriptionwillswitchedoff.


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SOMETIMEStherepressorproteinintheabsenceoflactosewillbindtothe operator (block RNAPs transcription ) and there is (CAP CAMPCOMPLEX)bindtotheCAPsitetoincreaseRNAPstranscription,,sowehave an activator and inhibitor for transcription , which one will win(repressorproteinVSCAMPCAPCOMPLEX)??

)Trpoperon(tryptophanoperon


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-Bacteriacan'tsurviveintheabsenceoftryptophan(bacteriamustsynthesistryptophan)

-Synthesisoftryptophanconsistofalongpathwaywhichconsistofmanyenzymesthatarecomingfrommanygenes(genes:TRPL/TRPE/TRPD/TRPC/TRPB/TRPA),allthesegeneswillproduceenzymesinpolycystronicmRNAthatwillparticipatetosynthesizetryptophan,Allthesegenesareunderthecontrolofonepromoter.

-Thesamethingasinlacoperon,thereisanoperatorregionoverlappingwiththepromoterandthereisarepressorgenesthatwillproducetrprepressor,ifthebacteriacelldoesn’thaveanytryptophan,itneedtoswitchontrpoperontosynthesizetryptophan.

-IfE.Colidoesn’thaveanytryptophan(NoenoughtryptophaninthemediaforE.COLItogrow),doesE.colineedtoswitchontryptophanoperonornot?

Logically,E.coliisverysmart,ithastobeveryactivetosynthesistryptophan,ithastoswitchontheoperontoproduceallenzymesfortryptophansynthesis.


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Iftryptophanisplenty,E.colidoesn’trequiretoswitchontheoperon(doesn’twanttowasteenergy).

-HowthissmartE.colidecidewhatisthemechanism?

Whenthebacteriahasalotoftryptophan,itwillbindtotherepressorthentherepressorwillbindtotheoperator,thuspreventingRNAPsfrombindingtoitspromoterthusswitchingofftheoperon.

Butintheabsenceoftryptophan,repressorproteinisn’tfindinganytryptophantobindwithit,soitcantbindtotheoperatorsoRNAPsbindseasilytoitspromoterswitchingonthetryptophanoperon.

-HowE.colicontroltheexpressionoftrpoperon?(anothermechanism)

Attenuation mechanism : a leader polypeptide is used to detect and sense if there is a plenty or no tryptophan , if there is plenty of tryptophan E.coli will switch off the operon , but if there is no tryptophan , E.Coli will switch on the operon and synthesize the enzymes that are responsible for the synthesis of tryptophan

Switch off the operon .


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-Hairpin loop used to terminate transcription in the mechanism that isn’t require any protein (protein independent mechanism )

- trp L: found b/w promoter operator region and other structural genes is transcribed into mRNA that called leader mRNA(leader peptide) .

*** Ribosome which is translating mRNA(that produced from the transcription of trpL) producing leader peptide and it continue to translate the whole mRNA .

Leader peptide will tell the E.Coli if there is high level of tryptophan or not .

CASE 1 (HIGH LEVEL OF TRYPTOPHAN) : this condition logically speaking the operon must be switched off .

When leader peptide and ribosome reaches tryptophan codon ( region1 ), it will sense the media which have a lot of tryptophan , it will pass very fast on region 1 and region 2 preventing the base pairing between 1 & 2 , and let region 3 & 4 to base pair together (complementary )forming a hairpin loop ( as atermination signal ) and thus transcription will stop .

CASE 2 (LOW LEVEL OF TRYPTOPHAN ) the operon must be switched on .

When ribosome reaches tryptophan codon , it will stop and wait tryptophan to come and incorporate it to the growing polypeptide chain to use it in translation , but there is no enough tryptophan so it will wait and wait and wait , 3&4 will not form hairpin structure , but 2&3 will form antiterminator structure 'as a result of waiting' , and thus transcription will continue and translation continue producing all the genes required for tryptophan synthesis .

So in case 2 ( E.coli uses translation & transcription to control the expression of tryptophan genes ) .


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this is to remind you about the termination codon(3&4)non termination codon (2&3).

There is another mechanism to regulate the gene expression that is called Riboswitch which is involving a binding of a molecule that’ll regulate the transcription of the mRNA for many genes (polycystronic) .

As you see here in the absence of the molecule you have this tertiary structure of the mRNA , in the presence of this molecule the tertiary structure of the mRNA will be changed and a terminator


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will be formed so the transcription here will stop.

Again: the molecule could bind the mRNA translating many genes and that molecule once it binds it changes the structure and form the tertiary structure and the terminator so the transcription will stop so this is the riboswitch. (very smart mechanism 😎)

Now we’re going to talk about the gene expression in the eukaryotic

It’s more complicated to regulate the gene expression in the eukaryotes than the prokaryotes, why?! Simply because the genome is more complicated ; why?! Because of the chromosome structure and the chromatin structure (is highly packed with proteins the DNA wraps around it and thus genes are not exposed to transcription factors or to RNA polymerase to bind to them; so there must be a mechanism to unwrapped the gene to be transcribed and the unwrapping genes in the eukaryotic system is taking place by modifications of histones mainly by acetylation)

**When we are talking about the expression we are talking about transcription and translation but mainly it’s transcription.


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How will acetylation of histones help in increasing the expression?! Histones are positively charged and the DNA that they wrap around it is negatively charged now if you acetylate histone, you prevent the positive charges so no packaging and the DNA will be exposed to the transcription factors to come and bind with the RNA polymerase to start the transcription.

**The unpacked portion of the genome is called the Eukromatic regions (active transcription and expression) while the packed is called Heterokromatic regions (inactive transcription and expression).

**What is chromatin?! Proteins + DNA. (so when we remove the protein we will have an exposed DNA ready for transcription)

**Example of transcription factors: transcription factor 2d

Other things that present in eukaryotes but not prokaryotes are enhancers.

**What are enhancers?! Specific sequences of proteins locate far away maybe 1000 of kilo bases (1000 base pair) from the promoter that could bind to specific proteins in specific tissue to express specific genes and this why our cells are different from each other. They increase the RNA polymerase binding affinity.


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