分子生物学英文版.docx

分子生物学英文版.docx

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分子生物学英文版

Chapter3NucleicAcid

1.PhysicalandchemicalstructureofDNA

●Double-strandedhelix

●Majorgrooveandminorgroove

●Basepairing

●Thetwostrandsareantiparallel

●G+Ccontent（percentG+C）

●SatelliteDNA

SatelliteDNAconsistsofhighlyrepetitiveDNAandissocalledbecauserepetitionsofashortDNAsequencetendtoproduceadifferentfrequencyofthenucleotidesadenine,cytosine,guanineandthymine,andthushaveadifferentdensityfrombulkDNA-suchthattheyformasecondor'satellite'bandwhengenomicDNAisseparatedonadensitygradient.

2.AlternateDNAstructure

Twobaseshavebeenextrudedfrombasestackingatthejunction.Thewhitelinegoesfromphosphatetophosphatealongthechain.Oisshownred,Nblue,PyellowandCgrey.

3.CircularandsuperhelicalDNA

DNAcanalsoformadouble-stranded,covalently-closedcircle.Thesecircularmoleculesareoftencoiledintoasuperhelix,theformationofwhichiscatalyzedbyenzymescalledtopoisomerases.

4.DenaturationofDNA

Denaturation:

Atransitionfromthenativetothedenaturedstate

DNAdenaturation:

alsocalledDNAmelting,istheprocessbywhichdouble-strandedDNAunwindsandseparatesintosingle-strandedstrandsthroughthebreakingofhydrogenbondingbetweenthebases.

Hyperchromicity/Hyperchromiceffect:

thestrikingincreaseinabsorbanceofDNA（A260）causedbythedenaturationofthedouble-strandedDNAmolecule

Meltingtemperature（Tm）:

thetemperatureatwhichhalfoftheDNAstrandsareinthedouble-helicalstateandhalfaredenatured.Themeltingtemperaturedependsonboththelengthofthemolecule,andthespecificnucleotidesequencecompositionofthatmolecule.

FactorsAffectingTm

●G-Ccontentofsample

●reagentsthatincreasethesolubilityofthebases（anythingthatdisruptsH-bondsorbasestacking）

●Saltconcentration

●pH

●Length

5.Renaturation

Strandscanbeinducedtorenature（anneal）underproperconditions.Factorstoconsider:

●Temperature

●Saltconcentration

●DNAconcentration

●Time

RepetitiveSequences

●Unique:

SingleCopyGenes

●Slightlyrepetitive（2-10copies）

●Middlerepetitive（10-hundreds）

--Clustered

--Dispersed

●Highlyrepetitive（hundredstomillions）

--ShortsequencesinsatelliteDNA

--Sequencesofnormallengthincertaingenesthatexistinverylargenumbers

C-valueParadox

ThereisapparentlyalackofassociationbetweenC-value（theamountofDNApresentinthehaploidgenomeofdifferentorganisms）andthedegreeoforganismalcomplexityofvariousmulti-cellularorganisms.In1971,Thomasnamedthisphenomenon,“C-valueParadox”.

在每一种生物中其单倍体基因组的DNA总量是特异的，被称为C值（CValue）。

C值和生物结构或组成的复杂性不一致的现象称为C值悖论（C-valueparadox）。

6.Hybridization

Hybridization:

thetechniquewhereinrenaturedDNAisformedfromseparatesingle-strandedsamples.

Heteroduplexing:

renaturationcombinedwithelectronmicroscopyinaprocedureallowsthelocalizationofcommon,distinct,andmissingsequencesinDNA.

DNA-RNAhybridization（Northernhybridization）:

theuseoffilterhybridizationtodetectsequencecomplementaritybetweenasinglestrandofDNAandanRNAmolecule.

7.ThestructureofRNA

Types:

mRNA,tRNA,rRNA

Distinctions:

-ribosereplacesdeoxyribose;

-UreplacesT;

-Single-stranded

Conformation:

stem-looporhairpin

8.Hydrolysisofnucleicacid

ThephosphodiesterbondsofbothDNAandRNAcanbebrokenbyhydrolysiseitherchemicallyorenzymatically.

Ribozymes:

theRNAenzymes,areabletocleaveandformspecificphosphodiesterbondsinamanneranalogoustoproteinenzymes.

Chapter6Thegeneticmaterial

ThePathtotheWatsonandCrickModel

1928,Griffith,transformationinpneumococci（肺炎球菌）

1944,Avery,Griffith’stransformingprinciplewasDNA

1950,Chargaff,apatternintheamountsofthefourbases

1952,HersheyandChase,DNAisthegeneticmaterial

1953,Franklin,thex-raypictureofDNA

Chargaff’srule

IntheDNAofallspeciesexamined,A=T,G=C

Thetotalamountofpurines（A+G）=pyrimidines（T+C）inDNA

Therationof（A+T）/（G+C）variesfromspeciestospecies

DNApropertiesandfunctions

1.DNAhastheabilitytostoregeneticinformation,whichcanbeexpressedinthecellasneed.

2.Thisinformationcanbetransmittedtodaughtercellswithminimalerror.（Thisprocessrequirescomplexenzymesandrepairmechanisms.）

3.DNApossessesbothphysicalandchemicalstabilitysoinformationisnotlostoverlongperiodsoftime（years）.

4.DNAhasthepotentialforheritablechangewithoutmajorlossofparentalinformation.

DNA-geneticmaterial:

Double-strandedDNAhasevolvedasthegeneticmaterialbecauseitisespeciallywell-suitedforreplication,repair,occasionalchange,andlong-timestability.

Gene:

Genescontainalltheinformationforthesynthesisandfunctioningofcellularcomponents.

Transcription:

theprocessofsynthesizingRNAmoleculesfromaDNAtemplate.

Triplets/codons:

theRNAnucleotidesequenceisread（onribosomes）insequentialgroupsofthreebases.

Mutation:

theprocessbywhichabase-sequencechanges.

Thecentraldogma:

DNAmakesRNA,makesprotein.

chapter7DNAreplication

Semiconservativereplicationofdouble-strandedDNA

UntwistingofhighlycoiledDNAisrequiredforDNAreplication

TopoisomeraseTypeI:

•WorkaheadofreplicatingDNA

•Mechanism

–Makesacutinonestrand,passesotherstrandthroughit.Sealsgap.

–Result:

theDNAis“relaxed”somewhat

Gyrase--ATypeIITopoisomerase

–Introducesnegativesupercoils

–breaksbothstrands

–Sectionlocatedawayfromactualcutisthenpassedthroughcutsite.

InitiationofDNAreplication

•Replicaioninitiatedatspecificsites:

OriginofReplication（ori）

•TwoTypesofinitiation:

–Denovo–SynthesisinitiatedwithRNAprimers.Mostcommon.

–Covalentextension—synthesisofnewstrandasanextensionofanoldstrand（“RollingCircle”）.Limitedtocertainviruses.

DenovoInitiation

•BindingtoOriCbyDnaAprotein

•OpensStrands

•Replicationproceedsbidirectionally

CovalentextensioninitiationRollingCircle

UnwindingofDNAforreplication

Helicase:

⏹Breakshydrogenbondsandeliminateshydrophobicinteractions

⏹NeedsenergysuppliedbyATP

⏹EncodedbytheDnaBgeneinE.coli

Single-strandDNAbindingproteins（SSB）:

Bindtotheexposedstrands,coatthemandblockthere-annealingprocess.

Elongationofnewlysynthesizedstrands

1.Thepolymerizationreactionandthepolymerases

Enzyme:

polymeraseIII

Needed:

substrates,template,primer

Direction:

5’→3’

2.Correctingmismatchedbases

The5’-3’exonucleaseactivityofpolIatasingle-strandbreak（nick）canoccursimultaneouslywithpolymerization----nicktranslation.

DNApolymeraseIIIconsistsofmultiplesubunits

⏹PolIandpolIIIarebothinvolvedinE.coliDNAreplication.PolIIIisthemajorpolymerase.

⏹BothpolyIandpolyIIIpossessaproofreadingoreditingfunction（3’-5’exonucleaseactivity）.

⏹The5’-3’exonucleaseactivityofpolIatasingle-strandbreak（nick）canoccursimultaneouslywithpolymerization----nicktranslation.

⏹DNApolymeraseIIIconsistsofmultiplesubunits.

⏹Allknownpolymerasescanworkonlyinthe5’-P→3’-OHdirection.

PolIandpolIIIhavesomefeaturesincommon:

●5’-3’polymerizationactivity

Thefourdeoxynucleoside5’-triphosphates

Aprimerwithafree3’-OH

Atemplate

●3’-5’exonucleaseactivity

AntiparallelDNAstrandsanddiscontinuousreplication

⏹ThetwostrandsofDNAisantiparallelandthereplicationisdiscontinuoussynthesis.

⏹Aprimerisrequiredforchaininitiationandtwodifferentenzymes（RNApolymeraseandprimase）areknowntosynthesizeprimerRNAmolecules.

⏹DNAligasejoinsprecursorfragmentsandpolIaswellasRNaseHparticipatesintheremovalofprimer.

RNApolymerase:

initiationofleading-strandsynthesis

Primase:

synthesisofprimersforlagging-strand

Primosome:

helicase/primasecomplex

PolI:

removaloftheprimerandreplacementofDNA

DNAligase:

joiningthefragment（gapsealed）

ThecompleteDNAreplicationsystem

BidirectionalreplicationspeedsupDNAsynthesis

Replicationofeukaryoticchromosomes

1.Eukaryoteshavemoreandlargechromosomes.

2.Eukaryoticreplicationmayrequireasmuchas6-8hoursforcompletionversusthe40minutesneededbyE.coli.

3.Therearemultiple,ratherthanasingle,replicationoriginsalongeukaryoticchromosomes.Theyarespacedabout20kbapart.

4.EukaryoticDNAreplicationisattherateofabout10-100nucleotidespersecondasopposedtotheprokaryoticrateofabout1500nucleotidespersecond.

5.AtleastfivetypesofDNApolymeraseshavebeenfoundineukaryoticcells.

真生物DNA的复制有DNA聚合酶及多种蛋白质因子参与，DNA聚合酶也有多种类型。

其中DNAPolα及DNAPolδ在细胞核内DNA的复制中起主要作用。

DNAPolδ催化前导链及滞后链的合成，是主要负责DNA复制的酶。

DNAPolα的功能主要是引物合成。

DNAPolγ是线粒体中的复制酶。

Chapter8Transcription

1.EnzymaticsynthesisofRNA

E.ColiRNApolymerase

Holoenzyme:

coreenzyme:

α2ββ’ω

σfactor

（1）BindingofRNApoltoatemplateatspecificsite

（2）Initiation

（3）Chainelongation

（4）Chainterminationandrelease

2.Transcriptionsignals

Inprokaryotes,thepromoterconsistsoftwoshortsequencesat-10and-35positionsupstreamfromthetranscriptionstartsite.

●the-10element:

Pribnowbox,usuallyconsistsTATAAT,isabsolutelyessentialtostarttranscriptioninprokaryotes.

●the-35element:

usuallyconsistsofTTGACA.Itspresenceallowsaveryhightranscriptionrate.

Inprokaryotes:

Ineukaryotes:

Termination

TerminationofRNAsynthesisoccursatspecificbase-sequencesintheDNAmolecule,calledterminators.

•Intrinsicterminators:

rho-independentterminators,theterminationsequencesallowRNApolymerasetoterminateelongationspontaneously.

•rho-dependentterm