基因表達(dá)及其調(diào)控

GeneExpressionOverview

SalwaHassanTeama

1

GeneExpressionTheGeneStructureTranscriptionGeneticCodeandProteinSynthesisRegulationofGeneExpressionProkaryotesVsEukaryotesGeneExpressionAnalysis2ContentsTargetaudiencePhysicianassistant;Postgraduateinclinicalspecialties;Medicalstudents;Medicaltechnologist;Beginners;andForeverylaboratoryworkerandeveryonepassionforlearning3GeneExpressionTheprocessbywhichagene'sinformationisconvertedintothestructuresandfunctionsofacellbyaprocessofproducingabiologicallyfunctionalmoleculeofeitherproteinorRNA(geneproduct)ismade.Geneexpression

isassumedtobecontrolledatvariouspointsinthesequence

leadingtoproteinsynthesis.

4GeneStructure

Eukaryoticgenestructure:Mosteukaryoticgenesincontrasttotypicalbacterialgenes,thecodingsequences(exons)areinterruptedbynoncodingDNA(introns).Thegenemusthave(Exon;startsignals;stopsignals;regulatorycontrolelements).Theaveragegene7-10exonsspreadover10-16kbofDNA.56ProteinSynthesis:FourstagesTranscriptionRNAprocessingTranslationPost-translationprocessing78GeneExpressionTranscriptionSynthesisofanRNAthatiscomplementarytooneofthestrandsofDNA.TranslationRibosomesreadamessengerRNAandmakeproteinaccordingtoitsinstruction.9ProteinSynthesis10Transcription

11TranscriptionEnzymesRNApolymerase:Theenzymethatcontrolstranscriptionandischaracterizedby:SearchDNAforinitiationsite,ItunwindsashortstretchofdoublehelicalDNAtoproduceasingle-strandedDNAtemplate,Itselectsthecorrectribonucleotideandcatalyzestheformationofaphosphodiesterbond,Itdetectsterminationsignalswheretranscriptends.12EukaryoticRNApolymeraseshavedifferentrolesintranscription

PolymeraseInucleolusMakesalargeprecursortothemajorrRNA(5.8S,18Sand28SrRNAinvertebratesPolymeraseII

nucleoplasmSynthesizeshnRNAs,whichareprecursorstomRNAs.ItalsomakemostsmallnuclearRNAs(snRNAsPolymeraseIIINucleoplasmMakestheprecursorto5SrRNA,thetRNAsandseveralothersmallcellularandviralRNAs.EukaryoticPromoterConservedeukaryoticpromoterelementsConsensussequenceCAATboxGGCCAATCTTATAboxTATAAGCboxGGGCGGCAPsiteTAC14

EukaryoticPromoterliesupstreamofthegene.Thereareseveraldifferenttypesofpromoterfoundinhumangenome,withdifferentstructureanddifferentregulatorypropertiesclass/I/II/III.TranscriptionFactorsTranscriptionfactorsareproteinsthatbindto

DNAnearthestartoftranscriptionofagene.TranscriptionfactorseitherinhibitorassistRNApolymeraseininitiationandmaintenanceoftranscription.15TranscriptionFactors16EnhancersEnhancersarestretchesofbaseswithinDNA,about50to150basepairsinlength;theactivitiesofmanypromotersaregreatlyincreasedbyenhancers

whichcanexerttheirstimulatoryactionsoverdistancesofseveralthousandsbasepairs.17PreinitiationComplexThegeneraltranscriptionfactorscombinewithRNApolymerase

toformapreinitiationcomplexthatiscompetenttoinitiatetranscriptionassoonasnucleotidesareavailable.

Theassemblyofthepreinitiationcomplexoneachkindofeukaryoticpromoter(classIIpromotersrecognizedbyRNApolymeraseII)

beginswiththebindingofanassemblyfactortothepromoter.1819Source:/health/What-is-Gene-Expression.aspx20Initiation

ThepolymerasebindingcausestheunwindingoftheDNAdoublehelixwhichexposeatleast12basesonthetemplate.

ThisisfollowedbyinitiationofRNAsynthesisatthisstartingpoint.21Initiation

TheRNApolymerase

startsbuildingtheRNAchain;itassemblesribonucleotidestriphosphates:ATP;GTP;CTPandUTPintoastrandofRNA.Afterthefirstnucleotideisinplace,thepolymerasejoinsasecondnucleotidetothefirst,formingtheinitialphosphodiesterbondintheRNAchain.22Elongation

RNApolymerase

directsthesequentialbindingofriboncleotidestothegrowingRNAchaininthe5'-

3'direction.EachribonucleotideisinsertedintothegrowingRNAstrandfollowingtherulesofbasepairing.ThisprocessisrepeatedutillthedesiredRNAlengthissynthesized……..23Termination

Terminators

attheendofgenes;signaltermination.TheseworkinconjunctionwithRNApolymerasetoloosentheassociationbetweenRNAproductandDNAtemplate.TheresultisthattheRNAdissociatefromRNApolymeraseandDNAandsostoptranscription.TheproductisimmatureRNAorpremRNA(Primarytranscript).24TheprimaryproductofRNAtranscription;thehnRNAscontainbothintronicandexonicsequences.ThesehnRNAsareprocessedinthenucleustogivematuremRNAsthataretransportedtothecytoplasmwheretoparticipateinproteinsynthesis.2526RNAProcessing(Pre-mRNA→mRNA)CappingSplicing

AdditionofpolyAtail27RNAProcessingCappingThecapstructureisaddedtothe5'ofthenewlytranscribedmRNAprecursorinthenucleuspriortoprocessingandsubsequenttransportofthemRNAmoleculetothecytoplasm.Splicing:

StepbystepremovalofpremRNAandjoiningofremainingexons;ittakesplaceonaspecialstructurecalledspliceosomes.28RNAProcessingAdditionofpolyAtail:Synthesisofthepoly(A)tailinvolvescleavageofits3'endandthentheadditionofabout40-200adenineresiduestoformapoly(A)tail.2930AlternativeSplicingAlternativesplicing:

isaverycommonphenomenoninhighereukaryotes.Itisawaytogetmorethanoneproteinproductoutofthesamegeneandawaytocontrolgeneexpressionincells.3132Source:/Class/MLACourse/Modules/MolBioReview/alternative_splicing.htmlTheGeneticCodeThesequenceofcodonsinthemRNAdefinestheprimarystructureofthefinalprotein.ThreenucleotidesinmRNA(acodon)specifyoneaminoacidinaprotein.33TheGeneticCode34TheGeneticCodeThetripletsequenceofmRNAthatspecifycertainaminoacid.64differentcombinationofbases;61ofthemcodefor20aminoacids(AA);thelastthreecodon(UAG,UGA,UAA)donnotcodeforaminoacids;theyareterminationcodons.Degenerate

Morethanontripletcodonspecifythesameaminoacid.35TheGeneticCodeUnambiguousEachcodonspecifiesaparticularaminoacid,thecodonACGcodesfortheaminoacidthreonine,andonlythreonine.NonoverlappingThismeansthatsuccessivetripletsarereadinorder.Eachnucleotideispartofonlyonetripletcodon.36DNACodon

RNACodon

37

Translation

38TranslationTranslation

istheprocessbywhichribosomesreadthegeneticmessageinthemRNAandproduceaproteinproductaccordingtothemessage'sinstruction.39RequirementforTranslation

RibosomestRNAmRNAAminoacidsInitiationfactorsElongationfactorsTerminationfactorsAminoacyltRNAsynthetaseenzymes:Energysource:40RibosomesEukaryoticribosomesarelarger.Theyconsistoftwosubunits,whichcometogethertoforman80Sparticle;60Ssubunitholds(threerRNAs5S,5.8S,28Sandabout40proteins).40Ssubunitcontains(an18SrRNAandabout30proteins).41ThelargeribosomalsubunitcontainsthreetRNAbindingsites,designatedA,P,andE.

TheAsite

bindsanaminoacyl-tRNA(atRNAboundtoanaminoacid);Psite

bindsapeptidyl-tRNA(atRNAboundtothepeptidebeingsynthesized).The

Esite

bindsafreetRNAbeforeitexitstheribosome.42PreparatoryStepsforProteinSynthesis

First,aminoacyltRNAsynthetasejoinsaminoacidtotheirspecifictRNA.Second,ribosomesmustdissociateintosubunitsattheendofeachroundoftranslation.43Theproteinsynthesisoccurin3phases

Accurateandefficientinitiation

occurs;theribosomesbindstothemRNA,andthefirstaminoacidattachedtoitstRNA.Chain

elongation,

theribosomesaddsoneaminoacidatatimetothegrowingpolypeptidechain.Accurateandefficienttermination,

theribosomesreleasesthemRNAandthepolypeptide.44Initiation

Theinitiationphaseofproteinsynthesisrequiresover10eukaryoticInitiationFactors(eIFs):

Factorsareneededtorecognizethecapatthe5'endofanmRNAandbindingtothe40sribosomalsubunit.BindingtheinitiatorMet-tRNAiMet(methionyl-tRNA)tothe40Ssmallsubunitoftheribosome.45InitiationScanningtofindthestartcodonbybindingtothe5'capofthemRNAandscanningdownstreamuntiltheyfindthefirstAUG(initiationcodon).ThestartcodonmustbelocatedandpositionedcorrectlyinthePsiteoftheribosome,andtheinitiatortRNAmustbepositionedcorrectlyinthesamesite.OncethemRNAandinitiatortRNAarecorrectlybound,the60Slargesubunitbindstoform80sinitiationcomplexwithareleaseoftheeIFfactors.46Elongation

Transferofproperaminoacyl-tRNAfromcytoplasmtoA-siteofribosome;Peptidebondformation;PeptidyltransferaseformsapeptidebondbetweentheaminoacidinthePsite,andthenewlyarrivedaminoacyltRNAintheAsite.Thislengthensthepeptidebyoneaminoacids.47ElongationTranslocation;translocationofthenewpeptidylt-RNAwithitsmRNAcodonintheAsiteintothefreePsiteoccurs.NowtheAsiteisfreeforanothercycleofaminoacylt-RNAcodonrecognitionandelongation.EachtranslocationeventmovesmRNA,onecodonlengththroughtheribosomes.48Termination

Translationterminationrequiresspecificproteinfactorsidentifiedasreleasingfactors,RFsinE.coliandeRFsineukaryotes.Thesignalsforterminationarethesameinbothprokaryotesandeukaryotes.ThesesignalsareterminationcodonspresentinthemRNA.Thereare3terminationcodons,UAG,UAAandUGA.49Termination

Aftermultiplecyclesofelongationandpolymerizationofspecificaminoacidsintoproteinmolecules,anonsensecodon=terminationcodonofmRNAappearsintheAsite.Theisrecognizedasaterminalsignalbyeukaryoticreleasingfactors(eRF)whichcausethereleaseofthenewlysynthesizedproteinfromtheribosomalcomplex.50PolysomesMostmRNAaretranslatedbymorethanoneribosomeatatime;theresult,astructureinwhichmanyribosomestranslateamRNAintandem,iscalledapolysomes.51ControlofGeneExpressionTranscriptional

Posttranscriptional

Translational

Posttranslational

52ControlofGeneExpression53Controlofgeneexpressiondependsvariousfactorsincluding:

Chromosomalactivationordeactivation.Controlofinitiationoftranscription.ProcessingofRNA(e.g.splicing).ControlofRNAtransport.ControlofmRNAdegradation.Controlofinitiationoftranslation(onlyineukaryotes).Post-translationalmodifications.5455GeneExpressionAnalysisPolymeraseChainReactionQuantitativePCRMicroarray…….……56EukaryoticGeneExpression

Essentiallyallhumans'genescontainintrons.Anotableexceptionisthehistonegeneswhichareintronless.Eukaryotegenesarenotgroupedinoperons.Eacheukaryotegeneistranscribedseparately,withseparatetranscriptionalcontrolsoneachgene.EukaryoticmRNAismodifiedthroughRNAsplicing.EukaryoticmRNAisgenerallymonogenic(monocistronic);codeforonlyonepolypeptide.57EukaryoticGeneExpression

EukaryoticmRNAcontainnoShine-Dalgarnosequencetoshowtheribosomeswheretostarttranslating.Instead,mosteukaryoticmRNAhavecapsattheir5`endwhichdirectsinitiationfactorstobindandbeginsearchingforaninitiationcodon.EukaryoteshaveaseparateRNApolymeraseforeachtypeofRNA.Ineukaryotes,polysomesarefoundinthecytoplasm.EukaryoticproteinsynthesisinitiationbeginswithmethioninenotNformyl-methionine.58Prokaryoticvs.EukaryoticBacterialgeneticsaredifferent.Prokaryotegenesaregroupedinoperons.ProkaryoteshaveonetypeofRNApolymeraseforalltypesofRNA,mRNAisnotmodifiedTheexistenceofintronsinprokaryotesisextremelyrare.59Prokaryoticvs.EukaryoticToinitiatetranscriptioninbacteria,sigmafactorsbindtoRNApolymerases.RNApolymerases/sigmafactorscomplexcanthenbindtopromoterabout40deoxyribonucleotidebasespriortothecodingregionofthegene.Inprokaryotes,thenewlysynthesizedmRNAispolycistronic(polygenic)(codeformorethanonepolypeptidechain).Inprokaryotes,transcriptionofageneandtranslationoftheresultingmRNAoccursimultaneously.Somanypolysomesarefoundassociatedwithanactivegene.60GlossaryAllelesareformsofthesamegenewithsmalldifferencesintheirsequenceofDNAbases.Alternativesplicing:isaverycommonphenomenoninhighereukaryotes.Itisawaytogetmorethanoneproteinproductoutofthesamegeneandawaytocontrolgeneexpressionincells.Exon:asegmentofagenethatisrepresentedinthematureRNAproduct.IndividualexonsmaycontaincodingDNAand/ornoncodingDNA(untranslatedsequences).Bioinformatics

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istheapplicationof

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informationtechnologytothefieldof

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Introns(interveningsequence)(AnoncodingDNAsequence):InterveningstretchesofDNAthatseparateexons.Primarytranscript:Theinitialproductionofgenetranscriptioninthenucleus;anRNAcontainingcopiesofallexonsandintrons.RNAgeneornon-codingRNAgene:RNAmoleculethatisnottranslatedintoaprotein.NoncodingRNAgenesproducetranscriptsthatexerttheirfunctionwithouteverproducingproteins.Non-codingRNAgenesincludetransferRNA(tRNA)andribosomalRNA(rRNA),smallRNAssuchassnoRNAs,microRNAs,siRNAsandpiRNAsandlastlylongncRNAs.Enhancersandsilencers:areDNAelementsthatstimulateordepressthetranscriptionofassociatedgenes;theyrelyontissuespecificbindingproteinsfortheiractivities;sometimesaDNAelementscanacteitherasanenhancerorsilencerdependingonwhatisboundtoit.Activators:Additionalgene-specifictranscriptionfactorsthatcanbindtoenhancerandhelpintranscriptionactivation.Openreadingframe(ORF):Areadingframethatisuninterruptedbytranslationstopcodon(readingframethatcontainsastartcodonandthesubsequenttranslatedregion,butnostopcodon).Directionality:inmolecularbiology,referstotheend-to-endchemicalorientationofasinglestrandofnucleicacid.Thechemicalconventionofnamingcarbonatomsinthenucleotidesugar-ringnumericallygivesrisetoa5'endanda3'end("fiveprimeend"and"threeprimeend").Therelativepositionsofstructuresalongastrandofnucleicacid,includinggenes,transcriptionfactors,andpolymerasesareusuallynotedasbeingeitherupstream(towardsthe5'end)ordownstream(towardsthe3'end).ReverseTranscription:Someviruses(suchasHIV,thecauseofAIDS),havetheabilitytotranscribeRNAintoDNA.Pseudogenes.DNAsequencesthatcloselyresembleknowngenesbutarenonfunctional.More:/books/NBK7584/61ReferencesandFurtherReadingAliKhalifa.Appliedmolecularbiology;eds:(FathiTashandSannaEissa).109pages.Egypt.UniversityBookCenter.2002.AvailableinpapercopyfromthepublisherDanielH.Farkas.DNASimplified:TheHitchhiker'sGuidetoDNA.110pages.Washington,DC:AACCPress,1996,ISBN0-915274-84-1.AvailableinpapercopyfromthepublisherDanielP.Stites,AbbaT.Terr.BasicHumanImmunology:336Pages.Appleton&Lange(November1990).ISBN.0838505430.AvailableinpapercopyfromthepublisherInnis,DavidH.Gelfand,John