WelcomeEachofYoutoMyMolecularBiologyClassMolecularBiologyoftheGene,5/E

---Watsonetal.(2004)PartI:ChemistryandGeneticsPartII:MaintenanceoftheGenomePartIII:ExpressionoftheGenomePartIV:RegulationPartV:Methods3/08/05PartII:MaintenanceoftheGenomeDedicatedtothestructureofDNAandtheprocessesthatpropagate,maintainandalteritfromonecellgenerationtothenextMaintenanceoftheGenomeCh6:ThestructuresofDNAandRNACh7:Chromosomes,chromatinsandthenucleosomeCh8:ThereplicationofDNACh9:ThemutabilityandrepairofDNACh10:HomologousrecombinationatthemolecularlevelCh11:Site-specificrecombinationandtranspositionofDNA3/08/05CHAPTER6TheStructuresofDNAandRNA

HowdothestructuresofDNAandRNAaccountfortheirfunctions?OUTLINE1.DNAStructure2.DNATopology3.RNAStructureDNASTRUCTUREDNASTRUCTURE(1)DNAiscomposedofpolynucleotidechainsStructure:twistingaroundeachotherintheformofadoublehelix.SchematicmodelSpace-fillingmodelNucleoside&Nucleotide,thefundamentalbuildingblockofDNAglycosidicbondphosphoesterbondDNApolarity:isdefinedbytheasymmetryofthenucleotidesandthewaytheyarejoined.Phosphodiesterlinkages:repeating,sugar-phosphatebackboneofthepolynucleotidechainBasesinDNApurinespyrimidinesadenineguaninecytosinethymineN9N1DNASTRUCTURE(2)Eachbaseshasitspreferredtautomericform(RelatedtoCh9)Thetwostrandsofthedoublehelixareheldtogetherbybasepairinginanantiparallelorientation,Whichisastereochemical

consequenceofthewaythatadenineandthymine,andguanineandcytosine,pairwitheachother.(Relatedtoreplicationandtranscription)DNASTRUCTURE(3)TheTwoChainsoftheDoubleHelixHaveComplementarySequencesExample:Ifsequence5’-ATGTC-3’ononechain,theoppositechainmusthavethecomplementarysequence3’-TACAG-5’DNASTRUCTURE(4)Watson-CrickBasePairing(Relatedtoreplicationandtranscription)Thestrictnessoftherulesfor“Waston-Crick”pairingderivesfromthecomplementaritybothofshapeandofhydrogenbondingpropertiesbetweenadenineandthymineandbetweenguanineandcytosine.A:CincompatibilityHydrogenBondingIsImportantfortheSpecificityofBasePairingDNASTRUCTURE(5)Thehydrogenbondsbetweencomplementarybasescontributetothethermodynamicstabilityofthehelix(why?)andthespecificityofbasepairingStackinginteractionsbetweenbasessignificantlycontributetothestabilityofDNAdoublehelixThedoublehelixhasMinorandMajorgrooves(What&Why)DNASTRUCTURE(5)(SeetheStructuralTutorialofthischapterfordetails)Itisasimpleconsequenceofthegeometryofthebasepair.TheMajorgrooveisrichinchemicalinformation(Whatarethebiologicalrelevance?)DNASTRUCTURE(6)Theedgesofeachbasepairareexposedinthemajorandminorgrooves,creatingapatternofhydrogenbonddonorsandacceptorsandofvanderWaalssurfacesthatidentifiesthebasepair.A:H-bondacceptorsD:H-bonddonorsH:non-polarhydrogensM:methylgroupsThedoublehelixexistsinmultipleconformations.DNASTRUCTURE(7)TheBform(10bp/turn),whichisobservedathighhumidity,mostcloselycorrespondstotheaveragestructureofDNAunderphysiologicalconditionsAform(11bp/turn),whichobservedundertheconditionoflowhumidity,presentsincertainDNA/proteincomplexes.RNAdoublehelixadoptsasimilarconformation.DNAstrandscanseparate(denature)andreassociate(anneal)DNASTRUCTURE(8)KeytermstounderstandDenaturationHybridizationAnnealing/renatureAbsorbanceHyperchromicityTm(meltingpoint)DNATOPOLOGYDNATOPOLOGY(1)Structure(1):Linkingnumberisaninvarianttopologicalpropertyofcovalentlyclosed,circularDNA(cccDNA)Linkingnumberisthenumberoftimesonestrandhavetobepassedthroughtheotherstrandinorderforthetwostrandstobeentirelyseparatedfromeachother.SpeciesofcccDNAPlasmidandcircularbacterialchromosomesLinearDNAmoleculesofeukaryoticchromosomesduetotheirextremelength,entrainmentinchromatinandinteractionwithothercellularcomponents(Ch7)Structure(2):LinkingnumberiscomposedofTwistandWritheThelinkingnumberisthesumofthetwistandthewrithe.Twististhenumberoftimesonestrandcompletelywrapsaroundtheotherstrand.WritheisthenumberoftimesthatthelongaxisofthedoublehelicalDNAcrossesoveritselfin3-Dspace.DNATOPOLOGY(2)LocaldisruptionofbasepairsFunction(1):DNAincellsisnegativelysupercoiled;nucleosomesintroducesnegativesupercoilingineukaryotesNegativesupercoilsserveasastoreoffreeenergythataidsinprocessesthatrequirestrandseparation,suchasDNAreplicationandtranscription.StrandseparationcanbeaccomplishedmoreeasilyinnegativelysupercoiledDNAthaninrelaxedDNADNATOPOLOGY(3)Function(2):Topoisomerases(P115-119)Thebiologicalimportanceoftopoisomerase?Thefunctionaldifferenceofthetwotypesoftopoisomerases?Theworkingmechanismoftopoisomerase(Seetheanimationfordetail)DNATOPOLOGY(4)RNASTRUCTURERNASTRUCTURE(1)RNAcontainsriboseanduracilandisusuallysingle-strandedBiologicalrolesofRNARNAisthegeneticmaterialofsomevirusesRNAfunctionsastheintermediate(mRNA)betweenthegeneandtheprotein-synthesizingmachinery.RNAfunctionsasanadaptor(tRNA)betweenthecodonsinthemRNAandaminoacids.RNAservesasaregulatorymolecule,whichthroughsequencecomplementaritybindsto,andinterfereswiththetranslationofcertainmRNAs.SomeRNAsareenzymesthatcatalyzeessentialreactionsinthecell(RNasePribozyme,largerRNA,self-splicingintrons,etc).Structure(1):RNAchainsfoldbackonthemselvestoformlocalregionsofdoublehelixsimilartoA-formDNARNASTRUCTURE(2)hairpinbulgeloopRNAhelixarethebase-pairedsegmentsbetweenshortstretchesofcomplementarysequences,whichadoptoneofthevariousstem-loopstructuresSometetraloopsequencecanenhancethestabilityoftheRNAhelicalstructuresForexample,UUCGloopisunexpectedlystableduetothespecialbase-stackingintheloopPseudoknotsarecomplexstructureresultedfrombasepairingofdiscontiguousRNAsegmentsFigure6-32Pseudoknot.Non-Watson-CrickG:UbasepairsrepresentadditionalregularbasepairinginRNA,whichenrichedthecapacityforself-complementarityFigure6-33G:UbasepairThedoublehelicalstructureofRNAresemblestheA-formstructureofDNA.Theminorgrooveiswideandshallow,butofferslittlesequence-specificinformation.Themajorgrooveissonarrowanddeepthatitisnotveryaccessibletoaminoacidsidechainsfrominteractingproteins.ThusRNAstructureislesswellsuitedforsequence-specificinteractionswithproteinsStructure(2):RNAcanfoldupintocomplextertiarystructuresRNASTRUCTURE(3)RNAhasenormousrotationalfreedominthebackboneofitsnon-base-pairedregionsWhy?InteractionsinthetertiarystructureUnconventionalbasepairing,suchasbasetriples,base-backboneinteractionsProteinscanassisttheformationoftertiarystructuresbylargeRNAmoleculeThecrystalstructureofa23SribosmeFunction:SomeRNAsareenzymesRNASTRUCTURE(4)RibozymesareRNAmoleculesthatadoptcomplextertiarystructureandserveasbiologicalcatalysts.RNasePandself-splicingintronsareribozymesStructure&Function:ThehammerheadribozymecleavesRNAbyformationofa2’,3’cyclicphophateRNASTRUCTURE(5)SeeanimationfordetailHomework(ontheCD)SeetheanimationsforDNAtopology,Topoisomerase,aswellasRibozymeStructureandActivity.Answeringthequestionsin“applyingyourknowledge”isrequired.Playthestructuraltutorial“IntroductiontotheDNAstructure”tobetterunderstandDNAstructureFinishallthecriticalthinkingexerciseKeypointsforChapter6Definitions:topoisomerase,ribozyme,doublehelix,DNAdenaturation,Tm,linkingnumber,pseudoknot.WhatarethestructuraldifferencesbetweenDNAandRNA?HowthestructuralpropertiesofDNAandRNAdeterminetheird