chapter 5-1 -fundamental molecular genetic mechanisms · pdf file05.04.2017 ·...

Chapter5-1- FundamentalMolecularGeneticMechanisms

5.1StructureofNucleicAcids5.2TranscriptionofProtein-CodingGenesandFormationofFunctionalmRNA5.3TheDecodingofmRNAbytRNAs5.4StepwiseSynthesisofProteinsonRibosomes5.5DNAReplication5.6DNARepairandRecombination5.7Viruses:ParasitesoftheCellularGeneticSystem

Whyisitnecessary?

Geneticengineering

Basicunderstanding

Whatdoyouwanttodo?

Overviewoffourbasicmoleculargeneticprocesses.

Canyouexplainthispicture?

FundamentalMolecularGeneticMechanisms

5.1Structure ofNucleicAcids• DNAandRNAstructureandfunction• ProteininteractionaffectsDNAstructure• ThreetypesofRNAsfunctioninproteinsynthesis

Chemicaldirectionalityofanucleicacidstrand.

Wherearethe5’endand3’end?

Chemicaldirectionalityofanucleicacidstrand.

Howtogrowfrom5’to3’?

Nucleotidestructure

NucleotideWhichoneistheattacker?

Electronattacks!

5’

3’

Whichdirectionwillitbegrown?

1. 5’=>3’2. 3’=>5’

TheDNAdoublehelix.• In1953,JamesD.WatsonandFrancisH.C.CrickproposedDNAdouble-helicalstructure,basedonanalysisofDNAfiberx-raydiffractionpatternsgeneratedbyRosalindFranklinandMauriceWilkinsandChargaff’srevelationthatthepercentagesofA=TandG=C.

• (a)Space-fillingmodelofBDNA(themostcommonformofDNAincells)

• bases(lightshades)projectinwardfromthesugar-phosphatebackbones(darkredandblue)ofeachstrand

• themajorandminorgroovesarelinedbypotentialhydrogenbonddonorsandacceptors(yellow),whichcaninteractwithproteinsandothermolecules

• (b)ChemicalstructureofDNAdoublehelixshowsthetwosugar-phosphatebackbonesand2hydrogenbondsinA-Tand3hydrogenbondsinG-Cbasepairs.

5’to3’

1. Doublehelix2. Majorandminorgrooves3. 5’to3’4. A-T,G-C

Majorandminorgrooves

1

2

G-CA-T

bindingWhatkindofbond?

Doublehelix

https://www.youtube.com/watch?v=ZGHkHMoyC5I

ComparisonofA-FormandB-FormDNA.• (a)BformofDNA:about10.5basepairsperhelicalturn;stackedbasepairsare0.34nmapart

• (b)AformofDNA:11basepairsperturn,withamuchdeepermajorgrooveandamuchshallowerminorgroovethaninB-formDNA.

• RemovalofwaterchangesthecrystallographicstructurefromBtoA.

ashorter,morecompacthelicalstructurewhose basepairs arenotperpendiculartothehelix-axisasinB-DNA.

5’to3’

StabilityofDNAandRNA

• DNAismorestablethanRNAandthereforeabettercarrierofgeneticinformation.

• RNAislessstablebecausethe2ʹ-hydroxylgroupinRNA(absentfrom“deoxy”DNA)canactasanucleophile,attackingthephosphodiesterbondandbreakingthestrand.

• The2ʹ,3ʹcyclicmonophosphateproductisfurtherhydrolyzedtoamixtureof2ʹand3ʹmonophosphates.

Base-catalyzedhydrolysisofRNA.

StabilityofDNAandRNA

DNA-proteincomplex

ArepresentativeDNAbindingprotein.

TATAbindingprotein(TBP)

Howtoknowthesitefortranscriptionstart?

DNA

TranscriptionstartsiteTATA

TATAboxA TATA box is a DNA sequence that indicates where

a genetic sequence can be read and decoded.

The TATA box is named for its conserved DNA sequence, which is most commonly TATAAA. Many eukaryotic genes have a conserved TATA box located 25-35 base pairs before the transcription start site of a gene.

DNA

TATATranscriptionstartsite

TATAbindingproteinTFIIDineukaryote

InteractionwithaproteincanbendDNA.• TheconservedC-terminaldomainoftheTATAbox–bindingprotein(TBP)bindstotheminorgrooveofspecificDNAsequences richinAandT,untwistingandsharplybendingthedoublehelix.

• TranscriptionofmosteukaryoticgenesrequiresparticipationofTBP.

Likeclip!

ThinktherolesofDNA…

Whichkindofstructuralchangesareimportant?

1. Replication2. Transcription

DNAmelting

DNAstrandsunwindandseparate(“denature”/“melt”)duringreplicationandtranscriptionbybreakingthehydrogenbondsbetweenbase-pairedbases.

HowcanyoumeltthedsDNA?

• byraisingtemperature,whichbreaksthehydrogenbondsbetweenbase-pairedbases.

Hydrogenbond

HowtoeasilydetectthedsDNAandssDNA?dsDNAvs.ssDNA– UVabsorptiondifference

• DuplexDNAbasepairsabsorblessUVlightthantheunpairedbasesinsingle-strandedDNA;duplexmeltingcausesanincreaseinUVlightabsorption(hyperchromicity).

Vs.

260nm

260nm Morefreetoabsorb!

G·CcontentofDNAaffectsmeltingtemperature.

G-Cvs.A-T,whichoneismorecriticalformeltingbytemperature?

• ThegreatertheG+Cpercentage,thehighertheTm;breakingthethreeG-ChydrogenbondsrequiresmoreenergythanbreakingthetwoA-Thydrogenbonds.

Whencanyouusethisknowledge?

1. Understandingthebasicreplicationandtranscriptionprocesses2. DNAmanipulationtechniques

1. Cloning2. Subcloning3. PCR4. DNAcutting…

Ex)PCRrx andG-Ccontent

MacrostructureofDNA

CircularDNA

TopoisomeraseIrelievestorsionalstressonDNA.

Manylinear(eukaryotegenomes,viralDNAs)andcircular(bacterialgenomes,mitochondrialDNA)aresubjecttotorsionalstressdevelopedduringreplicationandtranscription

HowtoseparatetheDNAbyweight?

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Yes!Negativecharge!!!

HowtoseparatetheDNAbyweight?

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Yes!Negativecharge!!!

DNAgelelectrophoresis

Question:Whichonemovesfaster?

Super-coiledCircular

Secondary&tertiarystructureofRNA

RNAsecondaryandtertiarystructures.

• RNAhasahydroxylat2’,uracil baseinsteadofthymine,andusuallyissingle-stranded.

• RibozymeRNAshavecatalyticactivitybasedontertiarystructuresformedbybaseparing.

• (a)BasepairingbetweendistantcomplementarysegmentsofanRNAmoleculeformshairpins(5–10nucleotideloop),stem-loops(11–100sofnucleotideloop),andothersecondarystructures,includingpseudoknotsthatcontributetotertiarystructure.

• (b)Pseudoknot:threediagramsofthehumantelomeraseRNAcoredomain

• Left:Secondary-structure,withbase-pairednucleotidesingreenandblueandsingle-strandedregionsinred

• Middle:Sequence(coloredtocorrespondtothesecondary-structurediagramattheleft)

• Right:3Dstructuredeterminedby2D-NMR,showingpairedbasesandonlyatubeforthesugarphosphatebackbone(coloredtocorrespondtothediagramsatleft)

FundamentalMolecularGeneticMechanisms

5.2TranscriptionofProtein-CodingGenesandFormationofFunctionalmRNA• DNAtranscription• Prokaryoticoperongeneorganizationandregulation• Eukaryoticgeneorganizationandregulation

WhichdirectionforRNAsynthesis?

RNAissynthesized5ʹ→3ʹ.

• OnegeneDNAstrandistemplatefortranscriptionofanRNAbypairingcomplementarybases.

RNApolymerasecatalyzesphosphodiesterbondformationbetweenthe3ʹoxygenofthegrowingstrandandtheαphosphateofacorrectlybase-pairedrNTP

Nowit’stimetothinkthestepsoftranscription!

dsDNA

RNA

ComplementarybindingfortranscriptionHowisitpossiblehere?

• Initiation:RNApolymerasemeltsDNAduplextoformatranscriptionbubbleandbeginpolymerizingribonucleotides(rNTPs)atthestartsite.

Threestagesintranscription.

Thinkthedirections…

dsDNA

RNA

5’3’

3’5’

5’3’ sense

Protein

Antisense

sense

• Elongation:Polymeraseadvances3’–5’downtemplatestrandpolymerizingonerNTP atatimeontothe3’endofgrowingRNA.The5ʹendoftheRNAstrandisdisplacedfromthetemplateDNAandexitsthroughachannelintheRNApolymerase.[SeparatedstrandsintheregionbehindthemovingRNApolymerase-transcriptionbubblereassociate intoadoublehelix.]

Threestagesintranscription.

Terminationisalsoimportant!

ssDNA

RNA

ssDNA

Stuckhere??!?!?

• Termination:RNApolymerasedissociatesfromthetemplateDNAataspecificterminationsequence(stopsite).

Threestagesintranscription.

BacterialRNApolymerase.

• RNApolymerasesofbacteria,archaea,andeukaryoticcells aresimilarinstructureandfunction.

• Composedoftworelatedlargesubunits(βʹandβ),twocopiesofasmallersubunit(α),andonecopyofafifthsubunit(ω)thatisimportantinpolymeraseassemblyandstabilitybutisnotessentialfortranscription,plusothersmallersubunitsinarchaeaandeukaryotes.

• PolymerasebendstranscriptionbubbletemplateDNA.

Bacterialtranscription

https://www.youtube.com/watch?v=1b-bRVgqof0

Howdoesthetranscriptionlook?

EukaryoteProkaryote

Youcansee…

Nascent pre-mRNAs.The RNA processing machinery is contained inside structures called "terminal knobs" in nascent RNA transcripts. Terminal knobs are visible in this electron micrograph of chromatin spreads from yeast.

Eukaryotictranscriptioninyeast

Geneorganizationinprokaryotesandineukaryotes.

• (a)Bacterial(E.coli)tryptophan(trp)operon:• acontinuouschromosomesegmentcontainingfivegenes(blue)thatencodetheenzymesnecessaryforthe

stepwisesynthesisoftryptophan,intheordertheenzymesfunctioninthetryptophansynthesispathway• theentireoperonistranscribedfromonepromoterintoonelongcontinuoustrpmRNA(red)• mRNAtranslationbeginsatfivedifferentstartsites,yieldingfiveproteins(green)

• (b)Thefivegenesencodingtheenzymesrequiredfortryptophansynthesisinbaker’syeast(Saccharomycescerevisiae):• onfourdifferentchromosomes• eachgeneistranscribedfromitsownpromotertoyieldaprimarytranscriptthatisprocessedintoafunctional

mRNAencodingasingleprotein

Geneorganizationinprokaryotesandineukaryotes.

• (a)Bacterial(E.coli)tryptophan(trp)operon:• acontinuouschromosomesegmentcontainingfivegenes(blue)thatencodetheenzymesnecessaryforthe

stepwisesynthesisoftryptophan,intheordertheenzymesfunctioninthetryptophansynthesispathway• theentireoperonistranscribedfromonepromoterintoonelongcontinuoustrpmRNA(red)• mRNAtranslationbeginsatfivedifferentstartsites,yieldingfiveproteins(green)

• (b)Thefivegenesencodingtheenzymesrequiredfortryptophansynthesisinbaker’syeast(Saccharomycescerevisiae):• onfourdifferentchromosomes• eachgeneistranscribedfromitsownpromotertoyieldaprimarytranscriptthatisprocessedintoafunctional

mRNAencodingasingleprotein

OperonIn genetics,an operon isafunctioningunitofgenomicDNAcontainingaclusterof genes underthecontrolofasingle promoter.

Thegenesare transcribed togetherintoan mRNA strandandeither translated togetherinthecytoplasm

TryptophanoperoninE.Coli

Geneorganizationinprokaryotesandineukaryotes.

• (a)Bacterial(E.coli)tryptophan(trp)operon:• acontinuouschromosomesegmentcontainingfivegenes(blue)thatencodetheenzymesnecessaryforthe

stepwisesynthesisoftryptophan,intheordertheenzymesfunctioninthetryptophansynthesispathway• theentireoperonistranscribedfromonepromoterintoonelongcontinuoustrpmRNA(red)• mRNAtranslationbeginsatfivedifferentstartsites,yieldingfiveproteins(green)

• (b)Thefivegenesencodingtheenzymesrequiredfortryptophansynthesisinbaker’syeast(Saccharomycescerevisiae):• onfourdifferentchromosomes• eachgeneistranscribedfromitsownpromotertoyieldaprimarytranscriptthatisprocessedintoafunctional

mRNAencodingasingleprotein

Structureofthe5ʹmethylatedcap.

• EukaryoticprecursormRNAsareprocessedatboth5’and3’endstoformfunctionalmRNAs.

• 5ʹmethylatedcap• addedbyenzymesas5’endemergesfromRNApolymerase

• protectsmRNAfromenzymaticdegradation,assistsexporttothecytoplasm,andisboundbyaproteinfactorrequiredtobegintranslationbyaribosomeinthecytoplasm.

• 5ʹ→5ʹlinkageof7-methylguanylatetotheinitialnucleotideofthemRNAmoleculeandmethylgroupadditiontothe2ʹhydroxyloftheriboseofbase1occurinallanimalandhigherplantcells

• yeastslackthemethylgrouponbase1• vertebratecellsalsomethylatethebase2ribose

3’modification&splicing

• RNAprocessingproducesfunctionalmRNAineukaryotes.

• 5ʹcap:addedduringformationoftheprimaryRNAtranscript.

• PolyAtail:• primarytranscriptiscleavedataspecificsitedownstreamofthetranslationSTOPcodonandmultiple(100–200)Aresidues(notencodedbythegeneDNAtemplate)areaddedenzymaticallybypoly(A)polymerase

• poly(A)tailstabilizesmRNAsinthenucleusandcytoplasmandisinvolvedinmRNAtranslation

• Splicing:removesintronsandjoinsexons

Alternativesplicing

• AlternativeRNAsplicingincreasesthenumberofproteinisoformsexpressedfromasingleeukaryoticgene.

• Fibronectinisamultifunctionalextracellularmatrixproteinthatissecretedfromfibroblasts(connectivetissuecells)andhepatocytes(livercells).

DNAtoRNA,theninnextclass

Translation

Replication

Discussionwithfriends

1.Seetheleftgraphandexplainhowthedouble

strandedDNAchangesitsabsorptionatacertain

pointoftemperature.

2.ThesamesequencesofDNAswhichhavedifferent

structures,super-coiledandcircularmoveddifferent

differencesingelelectrophoresis.Howcanyou

measuretherightsize(basepairs)?(Hint:Pleasefind

restrictionenzyme).

3.Whatarethedifferencesbetweentheprokaryoticand

eukaryotictranscriptions?

Pleasereadthisarticleandanswerthequestion.

http://www.nature.com/scitable/topicpage/rna-

transcription-by-rna-polymerase-prokaryotes-vs-961

4.RNAislessstablethanDNA.HowdoesmRNAkeepitsstability?Discussthemechanisms.

Etc (참고)

G·CcontentofDNAaffectsmeltingtemperature.

• DuplexDNAbasepairsabsorblessUVlightthantheunpairedbasesinsingle-strandedDNA;duplexmeltingcausesanincreaseinUVlightabsorption(hyperchromicity).