biochemistry chapter4.ppt

CHAPTER4DNAandRNA:Moleculesofheredity,DNA,RNA,andtheFlowofGeneticInformation,DNAandRNAarelonglinearpolymers线性聚合物,callednucleicacids核酸thatcarryinformationinaformthatcanbepassedfromonegenerationtothenext.(可以代代相传的)遗传信息携带者Thesemacromoleculesconsistofalargenumberoflinkednucleotides相互连接的核苷酸,eachcomposedofasugar,aphosphate,andabase.Sugarslinkedbyphosphatesformacommonbackbone,whereasthebasesvaryamongfourkinds.基本单位核苷酸(=核糖+磷酸+碱基)Geneticinformationisstoredinthesequenceofbasesalonganucleicacidchain.basesspecificpairs碱基对withoneanother(stabilizedbyhydrogenbonds)Thebasepairingresultsintheformationofadoublehelix碱基序列储存遗传信息Thesebasepairsprovideamechanismforcopyingthegeneticinformationinanexistingnucleicacidchaintoformanewchain.thegenesofallmoderncellsandmanyvirusesaremadeofDNA.DNAisreplicatedbytheactionofDNApolymerase聚合酶enzymes.DNA双螺旋结构使之能精确复制,传递遗传信息,Genesspecifythekindsofproteins,butDNAisnotthedirecttemplate模板forproteinsynthesis.基因决定蛋白Rather,thetemplatesforproteinsynthesisareRNA(ribonucleicacid)molecules.messengerRNA(mRNA)aretheinformation-carryingintermediatesinproteinsynthesis.transferRNA(tRNA)andribosomalRNA(rRNA),arepartoftheprotein-synthesizingmachinery.三种RNA协同构成蛋白合成机器AllformsofcellularRNAaresynthesizedbyRNApolymerasesthattakeinstructionsfromDNAtemplates.Thisprocessoftranscriptionisfollowedbytranslation,转录和翻译thesynthesisofproteinsaccordingtoinstructionsgivenbymRNAtemplates.Thus,theflowofgeneticinformation,orgeneexpression,innormalcellsis:GeneticcodedefinestherelationbetweenthesequenceofbasesinDNA(oritsmRNAtranscript)andthesequenceofaminoacidsinaprotein.Thecodeisnearlythesameinallorganisms:asequenceofthreebases,calledacodon,密码子specifiesanaminoacid.遗传密码决定碱基序列和AA序列间的关系,密码子确定氨基酸Thelastthemetobeconsideredistheinterruptedcharacterofmosteukaryotic真核genes,whicharemosaics镶嵌物ofnucleicacidsequencescalledintronsandexons.Botharetranscribed,butintronsarecutoutofnewlysynthesizedRNAmolecules,leavingmatureRNAmoleculeswithcontinuousexons.内含子和外显子,DNAconsistsof4kindsofbasesjoinedtoasugar-phosphatebackbone,DNAisapolymerofdNtunitsdNt(nucleotide)核苷酸consistsofnitrogenousbase含氮碱基(purine嘌呤:adenine腺,guanine鸟orpyrimidine嘧啶:thymine胸腺,cytosine胞),sugar(deoxyribose),andoneormorephosphategroupsNs(nucleoside)核苷consistsofpurineorpyrimidinebasebondedtosugar核苷=碱基+磷酸4NsinDNAare:deoxyadenosine,deoxyguanosine,deoxythymidine,anddeoxycytidineIndNs,N-9ofpurineorN-1ofpyrimidineisattatchedtoC-1ofdeoxyribose嘌呤9(嘧啶1)与(脱氧)核糖1之间形成糖苷键The3-hydroxylofthesugarmoietyofonedNtisjoinedtothe5-hydroxyloftheadjacentsugarbyaphosphodiesterbridge上一个dNt的3羟基与下一个dNt的5羟基之间以磷酸二酯键相连,Figure5.3.BackbonesofDNAandRNA.Thebackbonesofthesenucleicacidsareformedby3-to-5phosphodiesterlinkages.Asugarunitishighlightedinredandaphosphategroupinblue.,Figure5.4.PurinesandPyrimidines.Atomswithinbasesarenumberedwithoutprimes.UracilinsteadofthymineisusedinRNA.,transformationofPneumococci肺炎球菌byDNArevealedthatgenesaremadeofDNA,Apneumococcusisnormallysurroundedbyaslimy,glisteningpolysaccharidecapsule多糖荚膜thisouterlayerisessentialforthepathogenicity致病力ofthebacterium光滑型肺炎球菌外包一层粘性发光的多糖荚膜是其致病的必要成分，无荚膜的粗糙型因缺乏一种合成多糖所必要的酶而无法合成荚膜whichcausespneumoniainhumansandothersusceptiblemammalsmutantsdevoidofapolysaccharidecoatarenotpathogenicnormalbacteriumisreferredtoastheSform(菌落光滑)whereasmutantswithoutcapsulesarecalledRforms(lackinganenzymeneededforthesynthesisofcapsularpolysaccharide),anonpathogenicRmutantcouldbetransformedintothepathogenicSform供体(S)细胞的DNA进入受体(R)细胞而导入新的遗传信息,使非致病的R突变型转化成致病的S型miceisinjectedwithamixtureofliveRandheat-killedSpneumococci注入混合物(含热杀灭的S型和活的R型),S型的DNA将R型转化成了S型,所以(此混合物)是致病的thismixturewaslethaltothemice,whereaseitherliveRorheat-killedSpneumococcialonewerenotthebloodofthedeadmicecontainedliveSpneumococcitheheat-killedSpneumococcihadsomehowtransformedliveRintoliveS.thischangewaspermanent:thetransformedpneumococciyieldedpathogenicprogenyoftheSformthisRStransformationcanoccurinvitro体外,OswaldAveryandotherspublishedtheirdiscovery“anucleicacidofthedeoxyribosetypeisthefundamentalunitofthetransformingprincipleofpneumococcustype”,Theexperimentalbasisfortheirconclusionwas对纯化的,高度活性的(引起转化的)物质进行分析:elementalchemicalanalysisagreedcloselywiththatcalculatedforDNA化学构成optical,ultracentrifugal,diffusive扩散,andelectrophoreticpropertiesofthepurifiedmaterialwerelikethoseofDNA理化性质therewasnolossoftransformingactivityuponextractionofproteinorlipidthepolypeptide-cleavingenzymestrypsinandchymotrypsin胰凝乳蛋白酶didnotaffecttransformingactivity排除脂类和蛋白ribonuclease核糖核酸酶hadnoeffectonthetransformingprinciple排除RNAtransformingactivitywaslostfollowingtheadditionofdeoxyribonuclease确定是脱氧核糖核酸,thisworkisalandmarkinthedevelopmentofbiochemistryuntil1944,itwasgenerallyassumedthatchromosomalproteinscarrygeneticinformationandthatDNAplaysasecondaryroleitwasshatteredbythefindingthatpurifiedDNAhasgeneticspecificityFurthersupportforthegeneticroleofDNAcamefromthestudiesofavirusthatinfectsthebacteriumE.coliT2bacteriophage噬菌体consistsofacoreofDNAsurroundedbyaproteincoatDNA内芯+蛋白外壳,TheresultsoftheexperimentsofA.HersheyandM.Chasewere:mostofthephageDNAwasfoundinthebacteriamostofthephageproteinwasfoundinthesupernatant上清液theblendertreatment搅拌器(对附着在细菌表面的噬菌体施加强大的剪切力,从而使两者分离.)hadalmostnoeffectonthecompetence感受态oftheinfectedbacteriatoproduceprogenyvirus病毒外壳（蛋白）对（细菌）细胞内噬菌体(后代)生长没有作用,additionalexperimentsledtotheconclusion1.将病毒外壳(含35S,即“注射器”)与已被感染的细菌(即已“注射”了病毒DNA,)依靠剪切力分离.2.高速离心,使细菌沉淀(含32P),噬菌体蛋白外壳留在上清液中(含35S).aphysicalseparationofthephageT2intogeneticandnon-geneticpartsispossibleThesulfur-containingproteinofrestingphageparticlesisconfined限于toaprotectivecoatthatisresponsiblefortheadsorptionofbacteria附着细菌,andfunctionsasaninstrumentfortheinjectionofthephageDNAintothecell.TheDNAhassomefunction.inagivenspeciestheDNAcontentisthesameforallcellsthathaveadiploidsetofchromosomes二倍体.Haploid单倍体cellswerefoundtohavehalfasmuchDNA,ThediscoveryoftheDNAdoublehelixbyWastonandCrickrevolutionizedbiology,WastonandCricksmodelofDNA:1.twohelicalpolynucleotidechainsarecoiledaroundacommonaxis.Thechainsruninoppositedirections反平行双螺旋2.thePurandPyrbasesareontheinsideofthehelix,whereasthephosphateanddeoxyriboseunitsareontheoutside.Theplanesofthebasesareperpendiculartothehelixaxis.Thoseofthesugarsarenearlyatrightanglestothoseofthebases.碱基在内,磷酸核糖骨架在外,碱基平面与对称轴(即与骨架)垂直3.Thediameterofthehelixis20A.Adjacentbasesareseparatedby3.4Aalongthehelixaxisandrelatedbyarotationof36degrees.Hence,thehelicalstructurerepeatsafter10residuesoneachchain,thatis,atintervalsof34A.螺旋数据4.Thetwochainsareheldtogetherbyhydrogenbonds氢键betweenpairsofbases.AisalwayspairedwithT;GwithC.碱基互补5.Thesequenceofbasesalongapolynucleotidechainisnotrestrictedinanyway.Theprecisesequenceofbasescarriesthegeneticinformation.遗传信息是由碱基的精确顺序决定的,Figure5.11.Watson-CrickModelofDouble-HelicalDNA.Onepolynucleotidechainisshowninblueandtheotherinred.Thepurineandpyrimidinebasesareshowninlightercolorsthanthesugar-phosphatebackbone.(A)Axialview.Thestructurerepeatsalongthehelicalaxis(vertical)atintervalsof34,whichcorrespondsto10nucleotidesoneachchain.(B)Radialview,lookingdownthehelixaxis.,Figure5.12.StructuresoftheBasePairsProposedbyWatsonandCrick.,Figure5.13.AxialViewofDNA.Basepairsarestackednearlyoneontopofanotherinthedoublehelix.,themostimportantaspectoftheDNAdoublehelixisthespecificityofthepairingofthebases.A-T;G-C(becauseofstericandhydrogen-bondingfactors)（DNA结构和功能的精髓）碱基配对的特异性：由空间限制因素和氢键成键性质所决定thestericrestrictionisimposedbytheregularhelicalnatureofthesugar-phosphatebackboneofeachpolynucleotidechaintheglycosidicbonds糖苷键thatareattachedtoabondedpairofbasesareverynearly10.8AApurine-pyrimidinebasepairfitsperfectlyinthisspaceThebasepairingisfurtherrestrictedbyhydrogen-bondingrequirementsAcannotpairwithCbecausetherewouldbetwohydrogensnearoneofthebondingpositionsandnoneattheotherLikewise，GcannotpairwithTIncontrast,AformstwohydrogenbondswithT，GformsthreewithC,theearlierstudiesfoundthattheratiosofAtoTandofGtoCwerenearly1.0inallspeciesstudiedtheabove-mentionedistheBformofDNA.DNAcanassumedifferenthelicalforms，suchasA-DNA(由B型经脱水形成)andZ-DNA(左手双螺旋).Undermostphysiologicconditions,mostoftheDNAisintheWaston-CrickBform,Table5.1.Basecompositionsexperimentallydeterminedforavarietyoforganisms,thecomplementarychainsactastemplatesforeachotherinDNAreplication,DNAreplicationissemiconservative半保留,oneofthestrandsofeachdaughterDNAmoleculeisnewlysynthesized，whereastheotherispassedonunchangedfromtheparentDNAmoleculethedistributionof14Nand15Nwasrevealedbythenewlydevelopedtechniqueofdensity-gradientequilibriumsedimentation密度梯度沉降平衡Theabsenceof15NDNAindicatedthatparentalDNAwasnotpreservedasanintactunitonreplication.(byMeselsonandStahl)把细菌从15N介质转移到14N介质中后，每隔一段时间取样以提取其中的DNA，再用密度梯度技术分析。,thedoublehelixcanbereversiblymelted解链,DuringDNAreplicationandotherprocesses,thetwostrandsofthedoublehelixmustbeseparatedfromoneanother,atleastinalocalregion.解链才能复制thetwostrandsofaDNAhelixreadilycomeapartwhenthehydrogenbondsbetweenitspairedbasesaredisrupted氢键断裂byheatingasolutionofDNA加热orbyaddingacidoralkalitoionizeitsbases加酸碱使碱基离子化,theunwindingofthedoublehelixiscalledmelting熔化becauseitoccursabruptlyatacertaintemperaturethemeltingtemperature（Tm）isdefinedasthetemperatureatwhichhalfthehelicalstructureislost解链温度:螺旋-线圈转变的中点theabruptnessofthetransitionindicatesthattheDNAdoublehelixisahighlycooperative协同structure,heldtogetherbymanyreinforcingbondsitisstabilizedbythestackingofbases碱基堆积力aswellasbybasepairing配对碱基themeltingofDNAisreadilymonitoredbymeasuringitsabsorbanceoflightatwavelength260nm.测定光吸收值(的变化)判断是否已解链以确定DNA的”熔化”Theunstackingofthebasepairsresultsinincreasedabsorbance(Stackedbasesinnucleicacidsabsorblessultravioletlight紫外光thandounstackedbases),aneffectcalledhyperchromism增色性,themeltingtemperatureofaDNAmoleculedependsmarkedlyonitsbasecomposition.DNArichinG-CbasepairshaveahigherTmthanthosehavinganabundanceofA-Tbasepairs富含G-C对的DNA分子Tm高inaddition，adjacentG-CbasepairsinteractmorestronglywithoneanotherthandoadjacentA-Tbasepairs.HencetheA-T-richregionsofDNAarethefirsttomelt在同一DNA分子中,富含G-C对的区段先解链(“熔化”)thedoublehelixismeltedinvivobytheactionofspecificproteins（dnaAprotein）在体内不是靠高温而是由特定的功能蛋白(dnaAprotein)控制解链的separatedcomplementarystrandsofDNAspontaneouslyreassociatetoformadoublehelixwhenthetemperatureisloweredbelowTm.Thisrenaturation复性processissometimescalledannealing退火,DNAmoleculesareverylong,DNAmoleculesmustbeverylongtoencodethelargenumberofproteinspresentineventhesimplestcellsEcolichromosomeisasinglemoleculeofdouble-helicalDNAconsistingof4millionbasepairsItsmassis2.6x109daltons(4x106x600)ithasahighlyasymmetricshapewhentakenoutofthecelllength:14x106A(1.4mm);diameter:20AthelargestchromosomeofDrosophilamelanogaster(黑腹果蝇)containsasingleDNAmoleculeof6.2x107basepairs,whichhasalengthof2.1cm(6.2x107x3.4108)suchhighlyasymmetricmoleculesareverysusceptibletocleavagebyshearingforces,thefollowingcomparisonsemphasizetheremarkablelengthandasymmetryofDNAmoleculespolyomavirus多瘤病毒:5100basepairs,lengthof1.7mhemoglobin:diameterof65Aasroughlysphericalcollagen:lengthof3000A(oneofthelongestproteins),ManyDNAmoleculesarecircularandsupercoiled,intactDNAmoleculesfrommanysourcesarecircularthegene-linkagemapofE.coliiscircularthetermcircularreferstothecontinuityoftheDNAchain,nottoitsgeometricalformDNAmoleculesinvivonecessarilyhaveaverycompactshapeThelengthofE.colichromosomeisabout1000timesaslongasitsgreatestdiameterE.coli染色体长度(如完全伸展)是E.coli最大径向(真实)长度的1000倍,DNAfromtheT7bactreriophageislinear.DNAmoleculesofsomeviruses(bacteriophage嗜菌体)interconvertbetweenlinearandcircularforms.Thelinearformispresentinsidethevirusparticle,whereasthecircularformispresentinthehostcellAnewpropertyappearsintheconversionofalinearDNAduplexintoaclosedcircularmolecule.Theaxisofthedoublehelixcanitselfbetwistedtoformasuperhelix超螺旋,AcircularDNAwithoutanysuperhelicalturnsisknownasarelaxedmolecule.SupercoilingisbiologicallyimportantforasupercoiledDNAhasamorecompactshapethanitsrelaxedcounterpart(packagingofDNAinthecell)紧密的形状有利于在细胞中存在supercoilingaffectsthecapacityofthedoublehelixtounwind,andtherebyaffectsitsinteractionwithothermolecules紧密的超超螺旋结构影响双螺旋的解链,从而影响DNA与其它分子的相互作用,DNAisreplicatedbypolymerasesthattakeinstructionsfromtemplates,Thefullreplicationmachineryincellscomprisesmorethan20proteinsengagedinintricateandcoordinatedinterplay(thefirstisolatedenzyme)DNApolymeraseisa103-kdsinglepolypeptidechain.Itcatalyzesthestep-by-stepadditionofdNtunitstoaDNAchainItrequiresthefollowingcomponentstosynthesizeachainofDNA复制必需:酶以及(原料+引物+模板)dATP,dGTP,dTTP,dCTP(activatedprecursors),andMg2+aprimer引物chainwithafree3-OHgroupaDNAtemplate(single-ordoublestrandedwithoneofthechainsbrokenatoneormoresites),Figure5.21.PolymerizationReactionCatalyzedbyDNAPolymerases,Thechain-elongationreactioncatalyzedbyDNApolymeraseisanucleophilicattackofthe3-OHterminusoftheprimerontheinnermostphosphorusatomofadNTP引物3-OH端对加入的那个dNTP最靠里的(即)磷原子的亲核进攻ElongationoftheDNAchainproceedsinthe53directionDNApolymerasecatalyzestheformationofaphosphodiesterbondonlyifthebaseontheincomingnucleotideiscomplementarytothebaseonthetemplatestrandThus,DNApolymeraseisatemplate-directedenzyme以模板为指导的酶,AnotherstrikingfeatureofDNApolymeraseisthatitcorrectsmistakesinDNAbyremovingmismatchednucleotides(withnucleaseactivity)DNA聚合酶:1.是以模板为指导的酶2.它还具有纠错的功能,即能除去错配的(脱氧核糖)核苷酸ThesepropertiesofDNApolymerasecontributetotheremarkablyhighfidelity(保真度)ofDNAreplication,whichhasanerrorrateoflessthan108perbasepair,someviruseshavesingle-strandedDNAduringpartoftheircycle,NotallDNAisdouble-strandedDNAinX174,asmallvirusthatinfectsE.coli,issingle-strandeditsbaseratiosdonotconformtotherule:A=T;G=CitssolutionismuchlessviscousthanasolutionofthesameconcentrationofE.coliDNA.Itshydrodynamicpropertiesarelikethoseofarandomlycoiledpolymer(theDNAdoublehelixbehavesasaquiterigidrod)1.单链DNA粘度较低2.动力学性质与随机卷曲的聚合物相似,但不同于双链DNA(刚性杆状)3.它的碱基上的氨基易与甲醛反应,而双链DNA中碱基上的氨基不易与外来试剂接触.theaminogroupsofitsbasesreactreadilywithformaldehyde甲醛(thebasesindouble-helicalDNAarevirtuallyinaccessibletothisreagent),Thefindingofthissingle-strandedDNAraiseddoubtsconcerningtheuniversalityofthesemiconservativereplicativescheme半保留原则的普遍适用性.ButX174DNAissingle-strandedforonlyapartofthelifecycleofthevirus.infectedE.colicellscontainadouble-strandedformofX174DNAthisdouble-helicalDNAiscalledthereplicateformofX174DNA单链的X174噬菌体在感染(进入)E.coli后,以双链形式存在,即“复制型”thegeneralityoftheWatson-Crickschemeforreplicationwasreinforcedbythefindingofthisdouble-strandedviralDNAintermediate,thegeneofsomevirusesaremadeofRNA,GenesinallprokaryoticandeukaryoticorganismsaremadeofDNA.Inviruses,genesmadeofeitherDNAorRNARNA,likeDNA,isalong,unbranchedpolymerconsistingofNtjoinedby35phosphodiesterbonds,ThecovalentstructureofRNAdiffersfromthatofDNAin:thesugarunitsinRNAareribosesratherthandeoxyribose.Ribosecontainsa2-hydroxylgroupnotpresentindeoxyribose核糖-脱氧核糖(无2-羟基)oneofthefourmajorbasesinRNAisuracil(U)insteadofthymine(T)尿嘧啶胸腺嘧啶RNAmoleculescanbesingle-strandedordouble-strandedRNAcannotformadoublehelixoftheB-DNAtypebecauseofstericinterferencebythe2-hydroxylgroupsofitsriboseunitsRNA(核糖中的)2-羟基造成空间位阻,使得RNA不能形成B-DNA那样的双螺旋结构RNAcanadoptamodifieddouble-helicalform(likeA-DNA),TMV(TobaccoMosaicVirus烟草花叶病毒)isoneofthebest-characterizedRNAvirusesItconsistsofasinglestrandofRNAsurroundedbyaproteincoatTheproteincanbeseparatedfromtheRNAbytreatmentoftheviruswithphenol苯酚theisolatedviralRNAisinfective,whereastheviralproteinisnotAfterinfection,theprogenyvirusalwaysconsistedofRNAandproteincorrespondingtotheRNAintheinfectinghybridvirus.用杂合型病