遗传学新版课件 7

CHAPTER7GeneExpression:TheFlowOfGeneticInformationFromDNAViaRNAToProtein1.Geneexpression:

TheflowofgeneticinformationfromDNAviaRNAtoprotein-ThecentraldogmaAnmRNAIntermediaryATetrahymenacelllabeled3H-cytidine15min3H-cytidine12min;Nonradioativemedium88min

EvidencefromPhage-InfectedE.coli

SolSpiegelmanandcoworkers,1956

AnmRNAIntermediary:RapidswitchfromthetranscriptionofE.coli

genestophageT4genesinT4-infectedbacteria

AnmRNAIntermediary

DifferencesbetweenprokaryotesandeukaryotesOverviewTranscriptionTranslationProkaryotesEukaryotes

Geneexpression2.TheGeneticCode:

Howprecisegroupingsofthe4nucleotidesspecify20aminoacids

Agene’snucleotidesequenceiscolinearwithapolypeptide’samino-acidsequence(CharlesYanofsky)Gly(GGA),Arg(AGA)Glu(GAA)Gly(GGU),Cys(UGU)Glu(GAA)

Acodoniscomposedofthreenucleotides(FrancisCrickandSydneyBrenner,1961)

Triplet:Tripletreadingframe

andframeshiftmutation

IntragenicsuppressionandframeshiftmutationsBiochemicalmanipulationsrevealedwhichcodonsrepresentwhichaminoacids(MarshallNierenbergandHeinrichMatthaei,1961)NierenbergandPhilipLederin1965

Crackingthecode:UAG---amber(HarrisBernstein(amber),CharleySteinberg,DickEpstein,1960)UAA---ochreUGA---opalamberochreopal

TerminationCodonCorrelationofpolypeptidesinDNA,mRNA

andpolypeptide

TheGeneticCode:ASummary1.Thecodeconsistsoftripletcodons,eachofwhichspecifiesanaminoacid.2.Thecodonsarenonoverlapping.3.Thecodeincludesthreestop,ornonsense,codons:UAA,UAG,UGA.4.Thecodeisdegenerate,whichmeansthatinmanycasesmorethanonecodonspecifiesthesameaminoacid.5.Inreadingthetranscriptofagene,thecellularmachineryscansasinglestrandofmRNAfromafixedstartingpointthatestablishesareadingframe.6.Movingfromthe5’tothe3’endofanmRNA,…startingattheNterminusandmovingtowardtheCterminus7.Mutationsmaymodifythemessageencodedinasequenceofnucleotidesinthreeways:frameshiftmutation,missensemutation,Nonsensemutation8.Thegeneticcodeisalmost,butnotquite,universal.

OverlappinggenesinbacteriophageX174(Sanger,1977)

UsinggeneticstoverifythecodeCharlesYanofsky,invivoexperiment

3.Transcription:

RNApolymerasesynthesizesasingle-strandedRNAcopyofagene

ThreeRNAPolymerases/ThreeSetsofGenesCharacteristicsofthethreeRNApolymerasesofeukaryoteEnzymeLocationProductsSensitivitytoα-AmanitinRNApolymeraseINucleolusRibosomalRNAs,excluding5SrRNANosensitivityRNApolymeraseIINucleusNuclearPre-mRNACompletesensitivityRNApolymeraseIIINucleustRNAs,5SrRNA,andothersmallnuclearRNAsIntermediatesensitivityRNAprocessingineukaryotesAddingamethylatedcapatthe5’endandaPoly-Atailatthe3’end

InterruptedGenesinEukaryotes:ExonsandIntronsEarlyEvidenceforNoncodingSequencesinEukaryoticGenes

InterruptedGenes(splitgenes)R-loops:Numberofexonsandintrons

SplitGene:

Thehumandystrophingene(morethan80introns)DMD:Duchennemusculardystrophyexons:expressedregionsIntrons:interveningregions

RNAsplicing:

SplicesoutintronsandjoinsadjacentexonssnRNAs:UI,U2,U4,U5,U6100-300nucleotideslong

Spliceosome:

snRNAs,snRNPs,andtheSpliceosome(lariatstructure)

Pre-mRNASplicing:RemovalofIntronSequencesbyRNASplicing

intronintron

exon-GT……AG-exonexonintronexon

A64G73G100T100A100G100N

RNAPrecursorSplicing:

UniqueNucleaseandLigaseActivitiesAutocatalyticSplicing(self-splicing)

rRNAprecursors:Alternativesplicingandtrans-splicingDifferentmRNAscanbeproducedfromthesameprimarytranscript

AlternatesplicingoftheSxlgene

Trans-splicinginwheatmitochondria

AlteringtheInformationcontentofmRNAmolecules

RNAEditing:Editingofthemitochondrialcytochromeb

pre-mRNAinthetrypanosome

RNAEditing:HIVandreverse

transcription

BasepairingbetweenmRNAandtRNAsdirects

assemblyofapolypeptideontheribosome

tRNAmediatetheTransferofinformationfromnucleicacidtoprotein

4.Translation:BasepairingbetweenanmRNAcodonandatRNAanticodonWobble:

sometRNArecognizemorethanonecodonfortheaminoacidtheycarry

WobbleTheoryRibosomearethesitesofpolypeptidesynthesis

Synthesisandprocessingofthe30SrRNAprecursorinE.coliandthe45SrRNAprecursorinmammals

SynthesisandprocessingofrRNAs(a)(b)TheNobelLaureatesforChemistry2009文卡特拉曼·

拉马克里希南

VenkatramanRamakrishnan.UScitizen,SeniorScientistandGroupLeaderatStructuralStudiesDivision,MRCLaboratoryofMolecularBiology,Cambridge,UK.

托马斯·

施泰茨

ThomasA.Steitz,

UScitizen.SterlingProfessorofMolecularBiophysicsandBiochemistryandHowardHughesMedicalInstituteInvestigator,YaleUniversity,CT,USA

阿达·约纳特

AdaE.Yonath,Israelicitizen.DirectorofHelen&MiltonA.KimmelmanCenterforBiomolecularStructure&Assembly,atWeizmannInstituteofScience,Rehovot,Israel

DNATranscriptionandRNATranslation

RibosomeCrystalwithtRNAingrooves

70SribosomeofE.coli

TheNature,StructureandFunctionofRibosomes,EarlyCrystallographyIn1980,AdaYonathgeneratedthefirstthree-dimensionalcrystalsofaribosome’slargesubunit,and20yearslaterYonathgeneratedribosomeimagestodetermineeachatom’slocation.Yonathstabilizedcrystalsbyfreezingthem(liquidnitrogenat-196°C)andshecrystallizedribosomesfromotherresilientmicro-organisms.

RibosomesTheNature,StructureandFunctionofRibosomes,EarlyCrystallographyYonath‘sX-raycrystallographyyieldeda3-DribosomepictureoftheverystableribosomesfromGeobacillusstearothermophilus,(嗜热脂肪地芽孢杆菌)ahot-springinhabitant,abletosurvivetemperaturesupto75C.LaterYonathstudiedribosomesfromthesalt-lovingHaloarculamarismortui(嗜盐古细菌)fromtheDeadSea.Theseribosomesalsoareverystable,formgoodcrystalsandhelpedlocateeachatomintheX-raycrystal.Eventually,itwasrealizedthattheribosome’satomicstructurecouldbemapped,andmorescientistsjoinedintheracetodeterminethis-especially,ThomasSteitzandVenkatramanRamakrishnan.

RibosomesMolecularInsightsintoRibosomesX-RayDiffractionStudiesofProtein/RNAComplexesEveryribosomehastwosubunits,1smalland1large.Inhumans,thesmall,ribosomeunithas1largeRNAmoleculeandabout32proteins;thelargesubunithas3RNAmolecules,andabout46proteins.Eachsubunithasthousandsofnucleotidesandaminoacids,withhundredsofthousandsofatoms.Inribosomal,X-raydiffractiontherearemillionsofatomstoseeandstudy.

RibosomesMolecularInsightsintoRibosomesX-RayDiffractionStudiesofProtein/RNAComplexesIntheearly1990s,Yonath’scrystalshadclearpatternsofblackdots,helpfulfordeterminingatomiclocations,butlackingnecessaryphaseanglesforeachatomintheX-raycrystallography.Soakingthecrystalinmercury,taggedtheribosome'ssurfaceandpermittedcomparisonsofthecrystallinedottedpatterns,withandwithoutheavyatoms.Becauseribosomesaresolarge,toomanyheavyatomsattachedtotheribosome,anditwasdifficulttoimmediatelydeterminethephaseangles.

RibosomesMolecularInsightsintoRibosomesX-RayDiffractionStudiesofProtein/RNAComplexesSteitzusedribosomeimages,preparedbytheelectronmicroscopist,JoachimFrank,todeterminephaseangles.Usinginformationfromheavyatoms,thephaseanglewasdetermined.In1998,afterabout20yearsofwork,Steitzpublishedthefirstcrystalstructureoftheribosome’slargesubunitwitharesolutionof9Ångström(1Ångström=diameterofahydrogenatom).Individuala