分子生物學(xué)英文版課件：Chapter1 structure and function of nucleic acid

MolecularBiologyBasedontheNationalplanningtextbook，8thedition,2013

ContentsofmolecularbiologyNucleicacidandnucleotidesGeneexpression?SynthesisofDNA(replication)?SynthesisofRNA(transcription)?Synthesisofprotein(translation)RegulationofgeneexpressionGeneticengineeringSignaltransductionResearchHistoryofMolecularBiologyIn1869,F.Miescher:nucleinIn1928,FredGriffith:transformationIn1944,Averyandhiscolleges:DNA——geneticmaterialIn1953,WatsonandCrick:DNAdoublehelicalmodelIn1957,Kornberg:DNApolymeraseⅠIn1958,Crick:thecentraldogmaIn1961,JacobandMonod:operontheoryIn1966,Nirenberg、OchoaandKhorana:thegeneticcodonIn1970,TeminandBaltimore:reversetranscriptase

somesubstancefromtheheat-killedScellshadbeenpickedupbysomeoftheRcells,changingthemintovirulentStypecells.Griffithcoinedtheterm"transformation"todescribethisprocess.1928byFredGriffithIn1967-1970,R.YuanandH.O.Smith:RestrictionendonucleaseIn1975-1977,Sanger、MaxamandGilbert:DNAsequencingIn1990s,automaticDNAsequencingIn1981,Cech:rRNAselfsplicing－ribozymeIn1981,thefirsttransgenicmiceIn1985,Mullis:PCRIn1990,HGPlaunchedIn1990,ADAdeficiencygenetherapyIn1996,“Dolly”sheepIn1998,AndrewZ.FireandCraigC.Mello:RNAi(2006Nobelprize)

In2001,PublishingofthedraftversionoftheHumanGenomeTransgenicMouse,1982AchievementsofgeneengineeringTransgenicplant克隆羊“多莉”克隆羊“多莉”的研究者伊恩·維爾穆特（Wilmut）

NucleustransferringcloningBiochipHumanGenomeProjectDNARNAProteinTheCentralDogmaReplicationTranscriptionTranslationReversetranscriptionDNAandproteinChapter1structureandfunction

ofnucleicacidNucleicacidsarebiologicalinformationmacromoleculesClassification:Deoxyribonucleicacid,DNA:presentinnucleusandmitochondria;storeandcarrygeneticinformation.Ribonucleicacid,RNA:

presentincytoplasmandnucleus;involvedinexpressionofgeneticinformation.SectionIChemicalcompositionandprimarystructureofnucleicacid

Nucleotidesarebasiccomponentunitsofnucleicacid(RNA)(DNA)nucleotideBasePentosephosphateBasePurinepyrimidineAdenine,AGuanine,GCytosine,CThymine,TUracil,UPresentinbothDNAandRNA(OnlyinDNA)(OnlyinRNA)I.Chemicalcompositionofnucleotides酮式烯醇式互變異構(gòu)胺式

亞胺式互變異構(gòu)Pentose:riboseinRNA,deoxyriboseinDNATheprincipalderivativesofpurinesandpyrimidinesarenucleosidesandnucleotidesNucleoside=base(PurineoraPyrimidine)+pentose(riboseordeoxyribose)riboseribonucleoside2’-deoxyribosedeoxyribonucleosidepurinepurinenucleosidePyrimidinepyrimidinenucleosideBaseandribose(deoxyribose)arelinkedviaaglycosidicbond:ARCRNucleotide=nucleoside+phosphateor=Base(purineorpyrimidine)+pentose(riboseordeoxyribose)+phosphateriboseribonucleotide2’-deoxyribose deoxyribonucleotidePurinepurinenucleotidePyrimidinepyrimidinenucleotideNucleotidesarephosphorylatednucleosidesEsterbondNucleosidesandphosphatesarelinkedbyesterbond:EsterbondglycosidicbondTypesofribonucleotidesanddeoxyribonucleotides

DerivativesofnucleotideII.

primarystructureofnucleicacid

Theprimarystructureisthesequenceofnucleosidemonophosphatesfrom5’endto3’endinnucleicacid.(usuallywrittenasthesequenceofbases).

PhosphodiesterbondWrittenfrom5to3:basepentoseDNAA、G、C、TdeoxyriboseRNAA、G、C、UriboseThedifferencesbetweenDNAandRNA:SectionII

Spatial

structureandfunctionofDNA

Chargaff'sRules:

purines=pyrimidinesA=TG=CX-raydiffractionstudiesofFranklinandWilkinsHelixmoleculeDoublestrandsBackgroundsofpropositionofdoublehelixI.ThesecondarystructureofDNA—doublehelixmodel5′5′

3′

3′MinorgrooveMajorgrooveAntiparallelcomplementorydoublehelixstrand.Basepairing:A-TC-G

Pentose-phosphatebackboneoutsideandbasepairsinside.

Stabilizedbyhydrogenbondandbasestackingforce.geneticinformationconsistsintheDNAbasesequences1.

Twopolynucleotidechains,right-handeddoublehelix.2.ThetwostrandsofDNAareantiparallel.3.Pentose-phosphatebackboneoutsideandbasepairsinside4.Thesurfaceofthedoublehelixcontainstwogrooves:themajorandminorgrooves.5.Eachbaseishydrogenbondedtoabaseintheoppositestrandtoformabasepair.Theplanesofbasepairareperpendiculartoitsaxis.(ApairswithT;GpairswithC)6.thediameteris2.37nm,thepitchofaturnis3.54nm,10.5basepairsperturn,thedistancebetweentwobaseplanesis0.34nm.7.stabilizingforce:hydrogenbondandbasestackingforcePointsofDNAdoublehelicalmodel:DifferenttypesofDNA:ThedifferencesofthreetypesofDNA

A-DNAB-DNAZ-DNAhelixdiameterbp/perturnnm/perturnbackboneRight-handed2.55nm11bp2.53nmlinearRight-handed2.37nm10.5bp3.54nmlinearLeft-handed1.84nm12bp4.56nmzigzagTriplexDNAInthesetriplehelices,thethirdstrandremainsassociatedwithduplexDNAthroughnon-Watson-CrickinteractionsnowknownasHoogsteenpairing.

N6StructureofaG-quadruplex.Left:aG-tetrad.Right:anintramolecularG-quadruplex

II.SupercoiledstructureofDNAinprokaryotesrelaxedsupercoiledTelephonecord

III.nucleosomeineukaryotesnucleosomeNucleosome:

ThebindingofDNAbythehistonescreatesastructureunitofchromatin,thestructureunitiscallednucleosome.NucleosomelinkerNucleosomecore

HistoneH160bpofDNAOctamerofhistone(twomoleculeseachofH2A、H2B、H3、H4)150bpofDNAH1histone

IV.FunctionofDNA:*DNAiscarrierofgeneticinformation.Itcantransfergeneticinformationbyreplication.*Gene：Inmolecularterms,itisafunctionalsegmentofDNAthatcodesforpolypeptidechainorRNAmolecules.

*

Genomeisthetotalgeneticinformationofanorganism.Formostorganisms,itisthecompleteDNAsequence.ThechemicalnatureofRNAdiffersfromthatofDNAInRNA,thesugarisriboseratherthan2-deoxyriboseofDNA.Insteadofthymine,RNAcontainstheuracil.RNAexistsasasinglestrand,whereasDNAexistsasadouble-strandedhelicalmolecule.InRNA,guaninecontent≠cytosinecontent;adeninecontent≠u(mài)racilcontent.

SectionIII.StructureandfunctionofRNA

參與蛋白質(zhì)合成的三種主要RNA：

messengerRNAs(mRNAs,~5%oftotalRNA)TransferRNAs(tRNAs,~15%oftotalRNA)RibosomalRNAs(rRNAs,~80%oftotalRNA)

I.StructureandfunctionofmRNAThemostheterogeneousinsizeandstability.Serveastemplatesofproteinsynthesis.Uniquecharacteristics(ineukaryote):

capof5’-terminal(7-mGpppNm)tailof3’-terminal(polyA)

mRNAstructureinprokaryotes5’

3’StartcodonAUGstopcodonCodingregiontriplet5'untranslated

region3'untranslated

region?AGGA?Shine-Dalgarno(SD)sequence(Ribosomalbindingsite,RBS)SDsequence(RBS)isasequenceupstreamthestartcodeinprokaryoticmRNAthatcanbase-pairstoa?UCCU?sequenceatorverynearthe3'endof16SrRNA,therebybindingthemRNAandsmallribosomalsubunittoeachother.mRNAstructureineukaryotes5'm7Gppp

CapAAA???AAA3‘PolyAtailStartcodonAUGstopcodonCodingregiontriplet

CapRecognizedandboundbyCBP(cap-sitebindingprotein)duringinitiationoftranslationPolyAtailrecognizedandboundbyPAB(polyAbindingprotein)5'untranslatedregion3'untranslatedregion…..…CTCCTAGTACGATCGT….CTAGTCATTCGGTA…DNA5’cap..GAGGAUCAUGCUAGCA….GAUCAGUAAGCCAU...AAAAA…..mRNA

N-aa1aa2aa3……aaaa-C5’UTRCodingregion3’UTR3’polyAtailcappingenzyme5′，5′-GpppG-AATAAATTATTTGUGTCADNAmRNA7-mGpppNmcanbindwithCBPsFunctionsof5’capstructure(7-mGpppNm):Functionsof3’tail(polyA):polyAbindwithPABPCBP:capbindingprotein，帽結(jié)合蛋白PABP:poly(A)-bindingprotein,多聚腺苷酸結(jié)合蛋白-translocationofmRNAfromnucleartocytoplasm-regulatingtheinitiationoftranslation-increasingthestabilityofmRNATableforgeneticcodonhnRNASplicingTheroleofmessengerRNAs(mRNA):astemplate*ThisclassofRNAservesasthetemplatesusedtodeterminethesequenceofaminoacidsinproteinsynthesis.*InamRNA,beginningfromthefirstAUGthatissaidtobetheinitiationsignal(asainitiationcondon)everysubsequenttripletbasesisreadasacondonuntilreachingastopcondon(UAA,UGA,UAG).Openreadingframe(ORF)

5'CGGAAUG

GCA

GAG

UGG

CUA

UAAGCAUG

MetAlaGluTrpLeu

*Eachcodonspecifiesanaminoacid.ItisthecodonsequenceofamRNAthatdeterminestheaminoacidsequenceofaprotein.Varyinlengthfrom74to95nucleotides.Containsmorerarebases.Serveascarrierofaainthetranslation.

II.Structureandfunctionof

tRNArarebases(C-C-glycosidicbond)rarebasesFeaturesofsecondarystructureoftRNA:cloverleafFourloops:Dloop,anticodonloop,variableloop,andTψCloopFourarms:acceptorarm,Darm,TψCarm,andanticodonarmDDDSecondarystructure(cloverleaf)Tertiarystructure(inverse“L”)DDDTherolesoftransferRNAs(tRNAs):asanadaptertRNAsfunctionasanadaptermoleculeinvirtueofformingcovalentattachmentofindividualaminoacidcatalyzedbyaminoacyl-tRNAsynthetaseandrecognizingthecodonsequencesofthemRNAstoallowcorrectinsertionofaminoacidsintotheelongatingpolypeptidechain.subunitprokaryotes

（70S）eukaryotes

（80S）SproteinsrRNASproteinsrRNASmallersubunit3021kinds16S4033kinds18SLargersubunit5031kinds23S5S6050kinds28S5.8S5SComponentsofribosomesIII.Structureandfunctionof

rRNA真核生物18SrRNA原核生物翻譯過(guò)程中核糖體結(jié)構(gòu)模式:氨基酰位（aminoacylsite，A位）肽酰位（peptidylsite，P位）排出位（exitsite，E位）ThethreerolesofRNAinproteinsynthesis.MessengerRNA(mRNA)istranslatedintoproteinbythejointactionoftransferRNA(tRNA)andtheribosome,whichiscomposedofnumerousproteinsandtwomajorribosomalRNA(rRNA)moleculesⅣ.Othernon-codingRNA(ncRNA)

longnon-codingRNAandsmallnon-codingRNASnRNA(smallnuclearRNA)SnoRNA(smallnucleolarRNA)ScRNA(smallcytoplasmRNA)siRNA(smallinterferingRNA)

MicroRNAs

SmallcatalyticRNA:

Ribozymes,aretheRNAmoleculeswithcatalyticactivity.

Theactivityoftheseribozymesofteninvolvesthecleavageofanucleicacid.

RNAinterference(RNAi)被《Science》評(píng)為2002年最重大科技突破。RibozymeAnRNAmoleculewithcatalyticactivitySecondarystructureofribozyme------HammerheadstructureSectionⅣPhysicalandchemicalfeaturesofnucleicacidandapplications

Ⅰ.Generalfeaturesofnucleicacid:AcidityViscosityConformationAbsorbanceof260nm1.DNA或RNA的定量A260=1.0相當(dāng)于50μg/ml雙鏈DNA40μg/ml單鏈DNA（或RNA）20μg/ml寡核苷酸2.判斷核酸樣品的純度DNA純品:A260/A280=1.8RNA純品:A260/A280=2.0A260的應(yīng)用目錄Definition:

DNAdenaturationmeansthataDNAhaslostits’nativeconformationanddoublestrandDNAisseparatedtosinglestrandDNAbyexposedtoadestabilizingfactorsuchasheat,acid,alkali,ureaoramide.(whenhightemperatureisusedtodenatureDNA,theDNAissaidtobemelted).Ⅱ.DenaturationofDNADNAmeltingcurve:Tm

ismeltingtemperatureatwhichhalf(50%)ofDNAmoleculesaredenaturedTmisproportionalto(G+C)%ofDNAmoleculesTm=69.3+0.41×(G+C)%DNAlengthThefactorsofinfluencingTm:Double-strandedSinglestrandedDefinition:Hyperchromiceffect:DNArena