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1、Regulation of Gene Expression于敏代谢与分子医学教育部重点实验室1.Gene Expression and the Regulation of Gene Expression Gene ExpressionBiologicalprocesses,suchastranscription,andincaseofproteins,alsotranslation,thatyieldageneproduct.Ageneisexpressedwhenitsbiologicalproductispresentandactive.Geneexpressionisregulateda
2、tmultiplelevels.Type of gene expressionConstitutive expressionSome genes are essential and necessary for life,and therefore are continuously expressed,such as those enzymes involved in TAC.These genes are called housekeeping genes.Induction and repression The expression levels of some genes fluctuat
3、e in response to the external signals.Regulation of Gene ExpressionProkaryoticorganismsregulategeneexpressioninresponsetotheirenvironment.Eukaryoticcellsregulategeneexpressiontomaintainhomeostasisintheorganism.In space:Paddock S.W.(2001).BioTechniques 30:756-761.Eachcoloredstripeinthisflyembryoshows
4、theexpressionofadifferentgeneorsetofgenes.Thespatialregulationofthesegenesallowstheembryotobedividedupintodifferentregionsthatwillgiverisetothehead,theinternalorgans,theabdomen,etc.and in abundance:Clyde et al.(2003).Nature 426:849-853Notehowthegenewhoseexpressionisindicatedinbluevariesinabundancefr
5、omstrongexpression(boldarrow)toweak(thinarrow)withinitsexpressiondomain.Thesedifferencesinstrengthofgeneexpressionhaveimportantfunctionalconsequences.RNA polymeraseDNAmRNATranscriptional controlTranslational controlPost-translational controlProteinRibosomeRNA polymeraseOnset oftranscriptionLife span
6、(stability)of mRNATranslationrateProtein activationor inhibition(bychemicalmodification)Multi-level Regulation of gene expression Cis-acting elements isthe special DNA sequence that can affect the expression of its own gene.Features of Cis-acting elementsTrans-acting factorsDNAbindingdomainDBD-DNA结合
7、结构域Transcriptionactivatingdomain-转录活化结构域Domains of trans-acting factorsHelix-turn-helix motifsStructure:about 20 amino acids long2 short alpha helicies(7 9 amino acids long)DNA recognition helix(binds specific DNA sequence)Recognition helix and 2nd helix form 90 anglevery short turn(NOT a beta-turn)
8、Often glycine at start of the turn(helix breaker)Zinc-Finger MotifsCCHHZnCCHHZnCCHHZnSeveral subtypes(Cys4,Cys2-His2)Example:Cys2 His2 typeZinc does not interact with DNAUsually multiple zinc-fingers in a row At least some also bind RNAConsensus sequence:Y,F-X-C-X2-4-C-XXX-F-XXXXX-L-XX-H-X3-5-HBasic
9、 domainsLeucine zippers(bZip):BasicregionoftheproteinbindstoDNAMainlyactasdimersorothersometimesasothermultimersSpecialalpha-helicesallowformationofcoiled-coilstructures.Hydrophobicresidues(Leu)alignononesideofthehelix,NOTBindtoDNAExample:AP-1(JunandFos),C/EBPTheactivatingregionsaregroupedonthebasis
10、ofaminoacidscontent.Acidicdomains:containbothcriticalacidicaminoacidsandhydrophobicaa.yeastGal4yeastGal4Glutamine-richdomainsmammalianactivatorSP1Proline-richdomains:mammalianactivatorCTF1Transcription Activation Domains Characteristics of Gene Expression of ProkaryotesProkaryoticgenesarepolycistron
11、（多顺反子）systems,thatis,severalrelevantgenesareorganizedtogethertoformatranscriptionunit-operon（操纵子）.Prokaryoticcellsoftenrespondtotheirenvironmentbychangesingeneexpression.Genesinvolvedinthesamemetabolicpathwayareorganizedinoperons.Someoperonsareinducedwhenthemetabolicpathwayisneeded.Someoperonsarerep
12、ressedwhenthemetabolicpathwayisnolongerneeded.2.Regulation of ProkaryoticGene ExpressionProkaryotic RegulationControloftranscriptioninitiationcanbe:positive controlincreasestranscriptionwhenactivators(激活剂或激活蛋白激活剂或激活蛋白）bindDNAnegative controlreducestranscriptionwhenrepressors（阻遏剂或阻遏蛋白阻遏剂或阻遏蛋白）bindtoD
13、NAregulatoryregionscalledoperators（操作元件操作元件）Specificity factors:AlterthespecificityofRNApolymeraseExamples:s-factors(s70,s32)s s70s s32Heat shock geneHousekeeping geneHeat shock promoterStandard promoterFrancoisJacobandJacquesMonod(PasteurInstitute,Paris,France)Studiedtheorganizationandcontrolofthel
14、acoperoninE.coli.EarnedNobelPrizeinPhysiologyorMedicine1965.StudiedTwodifferenttypesofmutationsinthelacoperon:Mutationsinprotein-codinggenesequences.Mutationsinregulatorysequences.Discovery of OperonStructure of lac operon The DNA sequence that RNA-pol can bind to and initiate the transcription.Prom
15、oterThe DNA sequence adjacent to the structural genes that the repressor protein can bind to and prevent the transcription of structural genes.OperatorRegulation of ProkaryoticTranscriptionOperon typesInducibleoperon(可诱导型操纵子）Repressibleoperon（可阻遏型操纵子）Gene regulation of operonNegativeregulationPositi
16、veregulationPrimarytypeofcontrolofgeneexpression.Organization of lac operonlacIRegulatory genelacZlacYlacADNAmRNA-GalactosidasePermeaseTransacetylaseProteinStructural GenesPlacIPlacOlacrepressorRegulation of lac Operon Sequence of lac operonlac operon(TTTACA/TATGTT)is a weak promoter,and has a basal
17、 expression level.CAP(Catabolite gene activator protein,or cAMP Receptor Protein)binding site is at-60 region.CAP is a homodimer with binding ability to DNA and cAMP.Bacteria do not express these three enzymes when glucose is available.However,bacteria produce those enzymes if lactose is present and
18、 glucose is absent.Inducible expressionMetabolism of lactose Inducers异乳糖异乳糖 When lactose is absent,no lac gene is expressed.Situation 1lacI gene has its own promoter,and its expression can produce LacI repressor.The tetrameric Lac repressor binds to the lac operator site Olac.The binding blocks the
19、RNA-pol moving on DNA template,and no lacZ,lacY,and lacA are expressed.galactosidaseSituation 2When lactose is present,lacZ,lacY,and lacA genes are expressed.The galactosidase is weakly expressed(at the basal level).When lactose is present,it is converted to allolactose or galactose that binds to th
20、e repressor.The repressor can no longer bind to the operator,and lac gene can be expressed.Galactose,Allolastose and IPTG are referred to as inducer.Lactose is an indirect inducer.The lacZYA RNA transcript is very unstable and could be degraded quickly.Therefore,the synthesis of three enzymes will b
21、e cease under normal condition.Presence of lactosePositive control:CAP(catabolitegeneactivatorprotein)bindstopromoterwhenprokaryotemustuseanalternatefuel.activatedbycAMP.Ex.cAMPdependentonglucoseconcentration.Transcriptionoccursmorefrequently.Glucose inhibits the formation of cAMP.When glucose is pr
22、esent,cAMP is lower.Only after glucose is exhausted,cAMP becomes higher.The CAP-cAMP complex is formed,and this complex binds to the CAP binding site on lac operon.When glucose is present,the cAMP is low,no CAP-cAMP is formed and the expression of the lac operon is still low.Situation 3 When glucose
23、 is absent and lactose is present,the CAP-cAMP complex binds to the CAP site to activate the lac gene.Situation 4 Coordinate expressionNo glucoseGlucose Repressible OperonThetrpoperonisoneoftheconstitutivegenesexpressedatthebasallevel.Thestructuralgeneoftrpoperonencodes5enzymesusedforthesynthesisofT
24、rp.Thetrprepressorgenecanbeexpressed,butitdoesnotbindtotheoperator.WhenTrpismorethanenough,therepressorwillformacomplexwithTrp.Thecomplexbindstotheoperator,blockingthesynthesisofTrp.Trp operon Trp operonTrp repressorWhentryptophanlevelsarelow,repressordoesnotbindtooperatorandtryptophancanbemadeWhent
25、ryptophanlevelsarehigh,tryptophanbindstotherepressorandactivatesitanditbindstotheoperator(trp=corepressor)In addition to the repressor regulation,trp gene has a fine tuning mechanism called attenuation.The trp operon is regulated using attenuation mechanism at the translation level.Transcription Att
26、enuationIn prokaryotes,transcription and translation are physically coupledPrematureterminationoftranscriptionresultsinincompletemRNASynthesisofleadersequence=prematureterminationAttenuationoccursduetocouplingoftranscriptionandtranslationOrganization of trp operon Leader sequence Recall that a leade
27、r region(trpL)occurs between the operator and the trpE sequence.Within this leader is the attenuator sequence(att).att sequence contains a start codon,2 Trp codons,a stop codon,and four regions of sequence that can form three alternative secondary structures.Secondary structure SignalPaired region 1
28、-2pausePaired region 2-3anti-terminationPaired region 3-4termination Molecular model for attenuation Possible hairpins1/2 and 3/4 hairpin structure2/3 hairpinstructure Organization of the leader/attenuator trp operon sequence.Attenuation model in Trp starved cellsAttenuation model in Trp non-starved
29、 cellsGenerality of AttenuationSummary of Tryptophan operonRegulatedbytwocontrolmechanismsRepressorbindingtotheoperatorAttenuation:prematureterminationoftranscriptionAttenuation:Tryptophan OperonInvertedrepeatinmRNAoftrpoperonStemloopforms2conformations:3,4stemloopand2,3stemloop3,4stemloop=transcrip
30、tionpauseandreachterminationsignal2,3stemloop=donothaveaterminationsignalExcessTrpLeadersequencetranscribedandtranslatedfullyLeaderpolypeptideattachedtoribosomeCauses3,4stemloopconformation,whichsignalstranscriptionpausesiteandtranscriptionterminatedTrpconcentrationlowLeadersequencetranscribedandtra
31、nslateduntiltrp-richregionisreachedapauseoccursPausecauses2,3stemloopconformation,whichisnotaterminationsignalandtranscriptionoftrpgenescontinuesSummary of transcriptional Regulation Translation Initiation of Regulation Ribosomebindingsite(RBS)Shine-Dalgarno(SD)sequencingmRNAsecondarystructureInitia
32、tionalcodon Regulation of Rare CodonTranscriptional Regulation of Overlapping GeneTranslational RepressionPost-Transcriptional Regulation4.Time Regulation Differentsigmafactorsrecognizedifferentpromotersandthus,theavailabilityofsigmafactorscanregulatethetranscriptionofgenesassociatedwiththesepromote
33、rs.Time Regulation of Sigma FactorsEarlyMiddleLate phage-in Escherichia coli cell Upon infection,the phage has two mechanisms of action:Lysis(溶菌）溶菌）,inwhichthehostsgeneticmachineryisusedtoproduce100newphages,andthenthehostcellislysed(broken).Lysogeny（溶原）（溶原）,inwhichthephagechromosomeisintegratedinto
34、thehostsgenome.Thephagethendormantlyinfectsallprogeny,asitsgenome(calledtheprophage)isreplicatedwhenthehostdivides.Thephage“chooses”betweenthesetwomechanismsbasedona“reading”ofthehostsbehaviour.Ifthehostisgrowingwellthephagelysogenizesthehostandsubsequentlyinfectsallofitsprogeny.Ifthehostisnotgrowin
35、gwell(e.g.starving),thephagegrowslytically-anabandonshipresponse.This“decision”isbasedonageneticswitch.3.Regulation of Eukaryotic Gene expressionEvery cell has almost the same DNA and therefore the same genes.But different genes need to be“on”and“off”in different types of cells and different stages
36、of the cell growth.Therefore,gene expression must be regulated.embryobonelivermusclespermRegulation of Gene ExpressionProkaryoticorganismsregulategeneexpressioninresponsetotheirenvironment.Eukaryoticcellsregulategeneexpressiontomaintainhomeostasisintheorganism.Large genome:3 x 109 bps,35 000 genesMo
37、nocistronRepeated sequences:different lengths and different frequencies.Often inverted repeatsSplite genes:separated by introns and exons alternativelyFeatures of Eukaryotic Gene RegulationDNASignalGeneNUCLEUSChromatin modificationChromatinGene availablefor transcriptionExonIntronTailRNACapRNA proce
38、ssingPrimary transcriptmRNA in nucleusTransport to cytoplasmmRNA in cytoplasmTranslationCYTOPLASMDegradationof mRNAProtein processingPolypeptideActive proteinCellular functionTransport to cellulardestinationDegradationof proteinTranscriptionStages in gene expression that can be regulated in eukaryot
39、ic cellsRegulation of Chromatin Structure remodelingGeneswithinhighlypackedheterochromatinareusuallynotexpressedChemicalmodificationstohistonesandDNAofchromatininfluencebothchromatinstructureandgeneexpressionEukaryotic Chromosome StructureEukaryoticDNAispackagedintochromatin.Chromatinstructureisdire
40、ctlyrelatedtothecontrolofgeneexpression.ChromatinstructurebeginswiththeorganizationoftheDNAintonucleosomes.NucleosomesmayblockRNApolymeraseIIfromgainingaccesstopromoters.Organization/packaging of DNANucleus=5-10 m (0.01mm)Diploid genome=6.4x109 bp0.34nm/bpDNA=2 meters (2000 mm)Chromatin Remodeling I
41、neukaryotes,bindingofhistonestoformchromatingenerallyrepressesgeneexpression,makingspecificrepressorproteinsunnecessary.DNAnucleosomesmustbe“unwound”orremodeledtoallowtranscription.ChromatinremodelingcomplexesAcetylationorMethylationChromatin remodelingFigure 18-4Condensed chromatinDecondensed chrom
42、atinAcetyl group on histoneHistone ModificationsInhistone acetylation,acetylgroupsareattachedtopositivelychargedlysinesinhistonetailsThisprocessloosenschromatinstructure,therebypromotingtheinitiationoftranscriptionTheadditionofmethylgroups(methylation)cancondensechromatin;theadditionofphosphategroup
43、s(phosphorylation)nexttoamethylatedaminoacidcanloosenchromatinHistone ModificationsAdapted from Lund and Lohuizen Genes Dev 2004Histone Modification Status Correlates with Transcriptional ActivityGeneactivationcorrelatedwithH3-K9 acetylationGenesilencingassociatedwithH3-K9 methylationDNA Methylation
44、DNAmethylation,theadditionofmethylgroupstocertainbasesinDNA,isassociatedwithreducedtranscriptioninsomespeciesDNAmethylationcancauselong-terminactivationofgenesincellulardifferentiationInGenomic Imprinting,methylationregulatesexpressionofeitherthematernalorpaternalallelesofcertaingenesatthestartofdev
45、elopmentDNA Methylation Active transcriptionUnmethylatedCpGislandTFRNA polRepressed transcriptionMethylatedCpGislandTFRNA polCH3CH3CH3Epigenetic InheritanceAlthoughthechromatinmodificationsjustdiscusseddonotalterDNAsequence,theymaybepassedtofuturegenerationsofcellsTheinheritanceoftraitstransmittedby
46、mechanismsnotdirectlyinvolvingthenucleotidesequenceiscalledepigenetic inheritance 91More regulator bindings sites in multicellular organisms reflects the more extensive signal integration BacteriaYeastHumanEukaryotic TranscriptionGeneraltranscriptionfactorsbindtothepromoterregionofthegene.RNApolymer
47、aseIIthenbindstothepromotertobegintranscriptionatthestart site(+1).EnhancersareDNAsequencestowhichspecifictranscriptionfactors(activators)bindtoincreasetherateoftranscription.Transcriptional regulation of eukaryotic gene expressionControllingtheexpressionofeukaryoticgenesrequirestranscription factor
48、s.general transcription factorsarerequiredfortranscriptioninitiationrequiredforproperbindingofRNApolymerasetotheDNAspecific transcription factorsincreasetranscriptionincertaincellsorinresponsetosignals Cis-acting elements isthe special DNA sequence that can affect the expression of its own gene.Feat
49、ures of Cis-acting elementsPromoter Core promoter TATA-box,Initiator(Inr)Proximal elements of promoter CAAT-box,GC-box,CpG islands Typical Eukaryotic Gene,with Its Core Promoter and Proximal Control Region Sequence:TATAAAALocation:-25 -30 bpFunction:It is the binding site for TFII D,which is require
50、d for RNA polymerase binding.It controls the veracity and frequency of transcriptional initiation.TATA boxSequence:GCCAATLocation:-70 bpFunction:It is the binding site for CTF1(CAAT-binding transcription factor1)and C/EBP(enhancer binding protein).CAAT box Sequence:GGGCGGLocation:-30 -110 bpFunction