chapter 5-1 -fundamental molecular genetic mechanisms · pdf file05.04.2017 ·...
TRANSCRIPT
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?
-
-
--
--
- --
Yes!Negativecharge!!!
HowtoseparatetheDNAbyweight?
-
-
--
--
- --
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).