分子生物学Chapter 3 Nucleic Acids and Genomics.docx

分子生物学Chapter 3 Nucleic Acids and Genomics.docx

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分子生物学Chapter3NucleicAcidsandGenomics

Chapter3NucleicAcidsandGenomics

Reviewofnucleicacids

ThestructureofDNA,Deoxyribonucleicacid,wasfirstpublishedintheBritishjournalNaturein1953byJ.D.WatsonandF.H.Crick.However,theconstituentsofDNAhadbeenknownsincetheturnofthecentury.ThesimplemodelproposedbyWatsonandCrickimplementedX-raycrystallographyperformedbyM.H.F.WilkinsandR.Franklin.Additionally,workbyL.Paulingprovidedtherulesofbondingandtheelucidationofthealphahelixstructure.E.Chargraffhadprovidedthedataontheabundanceofthefournucleotides（Adenine,Thymine,Guanine,andCytosine）andtherelationshipsbetweenthemfromchemicalanalysis.E.ChargraffdeterminedthatdistributionofAandTwereproportional,andthatdistributionofGandCwerealsoproportionalsuggestingthecomplementaryarrangementofadenine-thymineandguanine-cytosine.SincethearrangementofDNAwascomplementary,thesequenceofonechainmustbecompatibletothesequenceoftheoppositechain.

FrancisCrick（left）,JimWatson（centre）,andLinusPauling（right）.

ImagesfromtheNobelPrizeFoundationwebsite

MauriceWilkinsshowedadiffractionpatternofDNAatascientificmeetinginNaplesin1951.ThiswasthefirstdiffractionpatternthatWatsonsawofDNAandithadadecisiveimpactinhisdecisiontostudyDNA.WatsonmovedtotheCavendishlaboratoryinCambridgewherehestruckuphisfamouscollaborationwithFrancisCrick.

Theirfirstattemptatamodelstructure,latein1951,waswrong-embarassinglysoforthematthetime,sincetheyhadarrangedforMauriceWilkinsandRosalindFranklintotravelupfromLondontoviewtheirstructure.Theirmodelwasatriplehelixinwhichthepolynucleotidebackboneswereplacedatthecentreofthestructurewiththebasespointingoutinsolution.But,thisarrangementofchainswaschemicallyimpossibleandoncethiswaspointedout,themodelfellapart.

X-RayfibrediffractionpattersofA-DNA（left）andB-DNA（right）.

ImagesfromtheMauriceWilkins1952NobelLectureattheNobelPrizeFoundationwebsite

Chapter3NucleicAcidsandGenomics

1.BuildingBlocks–Nucleotides

vAnucleotideiscomposedofthreeparts:

 pentose,baseandphosphategroup.

vA,C,GandT existinDNA;

vA,C,GandU existinRNA.

Bases:

BicyclicPurines:

Monocyclic

pyrimidine:

Nucleosides:

Thebasesarecovalentlyattachedtothe1’positionofapentosesugarring,toformanucleoside

Nucleotides:

Anucleotideisanucleosidewithoneormorephosphategroupsboundcovalentlytothe3’-,5’,or（inribonucleotidesonly）the2’-position.Inthecaseof5’-position,uptothreephosphatesmaybeattached.

BASES

NUCLEOSIDES

NUCLEOTIDES

Adenine（A）

Adenosine

Adenosine5’-triphosphate（ATP）

Deoxyadenosine

Deoxyadenosine5’-triphosphate（dATP）

Guanine（G）

Guanosine

Guanosine5’-triphosphate（GTP）

Deoxy-guanosine

Deoxy-guanosine5’-triphosphate（dGTP）

Cytosine（C）

Cytidine

Cytidine5’-triphosphate（CTP）

Deoxy-cytidine

Deoxy-cytidine5’-triphosphate（dCTP）

Uracil（U）

Uridine

Uridine5’-triphosphate（UTP）

Thymine（T）

Thymidine/

deoxythymidie

Thymidine/deoxythymidie

5’-triphosphate（dTTP）

Phosphodiesterbonds&primarysequence:

Primarysequence:

5’end:

notalwayshasattachedphosphategroups

3’end:

freehydroxyl

（-OH）group

2.DNAStructure

•WatsonandCrick,1953

•Thegeneticmaterialofallorganismsexceptforsomeviruses

•Thefoundationofthemolecularbiology

Basepairing

·TwoseparatestrandsAntiparellel（5’3’direction）

Complementary（sequence）

Basepairing:

hydrogenbondingthatholdstwostrandstogether

•Sugar-phosphatebackbones（negativelycharged）:

outside

•Plannerbases（stackoneabovetheother）:

inside

2.1DNA'sBForm,AFormandZForm

vInthisstructure,alsoknownastheBform,thehelixmakesaturnevery3.4nm,andthedistancebetweentwoneighboringbasepairsis0.34nm. Hence,thereareabout10pairsperturn. Theintertwinedstrandsmaketwogroovesofdifferentwidths,referredtoasthemajorgrooveandtheminorgroove,whichmayfacilitatebindingwithspecificproteins.

vThenormalright-handed"doublehelix"structureofDNA,alsoknownastheBform

v Inasolutionwithhighersaltconcentrationsorwithalcoholadded,theDNAstructuremaychangetoanAform,whichisstillright-handed,butevery2.3nmmakesaturnandthereare11basepairsperturn.

FeaturesoftheWatson-CrickmodelofB-DNA：

vItisanantiparalleldoublehelix.

vItisaright-handedhelix.

vThebase-pairsareperpendiculartotheaxisofthehelix.（Actually,theyareveryslightlytilted-atanangleof4degrees）

vTheaxisofthehelixpassesthroughthecentreofthebasepairs.

vEachbasepairisrotatedby36degreesfromtheadjacentbasepair.

vThebase-pairsarestacked0.34nmapartfromoneanother.

vThedoublehelixrepeatsevery3.4nm,i.e.thepitchofthedoublehelixis3.4nm.

vB-DNAhastwodistinctgrooves:

aMAJORgroove;and,aMINORgroove.Thesegroovesformasaconsequenceofthefactthatthebeta-glycosidicbondsofthetwobasesineachbasepairareattachedonthesameedge.However,becausetheaxisofthehelixpassesthroughthecentreofthebasepairs,bothgroovesaresimilarindepth.

TherealstructureofB-DNA

WatsonandCrick'sstructurewasajustamodel-butaprettygoodone.Nevertheless,ittooknearly30yearsbeforethestructuresofDNAwereresolvedatatomicresolution.

In1980,RichardDickersonandHoraceDrewsolvedthestructureofa12-merdouble-strandedself-complementaryoligonucleotidewiththefollowingsequence:

 5'-CGCGAATTCGCG-3'

TheirresultsshowedthatcrystalsofB-DNAhadastructureverysimilartothatproposedbyWatsonandCrick.Althoughtherewerenumeroussmallvariations,theoverallstructurewasasexpected.

∙Themoleculewasaright-handeddouble-helix.

∙Thebackbonechainswereantiparallel.

∙Thebasepairswereverynearlyperpendiculartothehelixaxis.

∙Thebasepairswerecentredonthehelixaxis.

∙Onaverageeachbasepairwasrotated35.6degreesfromtheadjacentbasepair.However,theindividualmeasuredrotations（twist）variedfromaslittleas28degreestoasmuchas42degrees.

Onaveragetheriseperbasepair（i.e.thedistancebetweenadjacentbasepairs）was0.34nm.However,therisebetweenindividualbasepairsvariedfrom0.274nmto0.435nm.

OneofthemoststrikingfeaturesofthestructuresolvedbyDickersonandDrewwasthatthereisconsiderablevariationinthestructureofindividualbasepairs.Manywerewerenotexactlyplanarbutwereslightlytwisted（propellortwist）.Thisfeature,aswellasthevariationsinthetwistanglesandtherisebetweenbasepairscanbeexplainedorunderstoodasresultingfromthecomplexinterplayofattractiveandrepulsiveforcesduetothedifferentchemicalpropertiesofthefourdistinctbaseswithinaDNAdoublehelix.ThedetailedstructureofaDNAmoleculeisactuallyinfluencedbythesequence.

A-DNA

 RecallthatthefirstfibrediffractionpicturestakenbyRosalindFranklinwereofadehydratedformofDNA,whichwenowknowasA-DNA.

AneasywaytorememberthebasicstructureofA-DNAistorememberthisfactaboutitswatercontent.InA-DNAthereisnowaterspine.Thebasepairs--whichneverthelessareformedwithcanonicalWatson-Crickhydrogenbonds--arepushedtowardstheminorgroove.Indoingso,thebasepairstiltto19degreesfromperpendiculartothehelixaxiswhichnolongerpassesthroughthecentreofeachbasepair.Theresultingminorgrooveisaboutaswideastheresultingmajorgroove.However,themajorgrooveisverydeepwhiletheminorgrooveisveryshallow.Inaddition,thesugarringchangesfromtheC2'-endoconformationfoundinB-DNAtoaC3'-endoconformation.ThischangeservestodistancethephosphatefromtheC2'hydroxylatomhencepreventingautocatalysis

ThefollowingimagesshowthestructureofA-DNA.

Notethefollowingabouttheseimages:

∙ThefirstimageshowstheA-DNAhelixfromtheside.Noticethatthebasepairsaretiltedwithrespecttotheaxisofthehelix.Mostofthebasepairsarenearlyplanar,but,aswithB-DNA,youcanseesomeexceptions.Youcanalsoseesomeindicationofpropellortwist.

∙Observethebasepairthatishalfwayalongthehelix.Yourviewofthisbasepairisessentiallyend-on.Fromthisperspective,youcanseethatthisbasepairisnotcentredonthehelixaxisbutisverymuchpushedtooneside.Themajorgroove（ontheright）isverydeep;theminorgroove（ontheleft）isveryshallow.

∙ThesecondimageshowstheA-DNAhelixfromoneend.Noticethatthereisessentiallyalarge"hole"inthecentrebecausealloftheatomsofthebasepairsarepushedouttothesides.

 AnotherDNAstructureiscalledtheZform,becauseitsbasesseemtozigzag. ZDNAisleft-handed. Oneturnspans4.6nm,comprising12basepairs. 

vTheDNAmoleculewithalternatingG-Csequencesinalcoholorhighsaltsolutiontendstohavesuchstructure.

Z-DNA

EversinceWatsonandCrickproposedtheirstructureforDNA,crystallographicproofwassought.In1979,AlexRichandhiscolleaguesatMITthoughttheyhadit!

Theysucceededincrystallizingtheself-complementaryhexanucleotide,CGCGCG.Theiranalysiswasasurprise.

Theircrystalsshowedthatthisparticularmoleculeadoptedaleft-handeddoublehelixundertheircrystallizationconditions.Thebaseshadadoptedasyn-conformationratherthantheusualanti-conformationwiththeresultthattherepeatingunitofthestructurewasadinucleotidebase