Chapter5-2- FundamentalMolecularGeneticMechanisms

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

Question:Howtoreaditfortranslation?

ssDNA

RNA

ssDNA

RNAPeptides…

FundamentalMolecularGeneticMechanisms

5.3TheDecodingofmRNAbytRNAs• DNA/RNAcodontripletcode• Codingsystem• tRNAstructureandfunction

ThreerolesofRNAinproteinsynthesis.

• ThreeRNAsinvolvedinproteinsynthesis:mRNA,tRNA,rRNA

• mRNA:nucleotidesequenceencodestheorderofaminoacidsaribosomeassemblesintopolypeptidechain

• tRNAs:• Eachaminoacidtypeiscovalently

boundtoasubsetoftRNAscontainingaspecificthree-nucleotideanticodon sequence.

• Eachanticodonbase-pairswithitscomplementarymRNAcodontopositiontheencodedaminoacidintheribosomeAsitewhereitiscovalentlylinkedtotheC-terminusofthegrowingpeptide.

• rRNA:• Ribosomesarecomposedof

numerousproteinsandthree(bacterial)orfour(eukaryotic)ribosomalRNA(rRNA)molecules(notshown).

• OneoftherRNAs catalyzespeptidebondformationbetweenincomingaa-tRNA amino-groupandthecarboxy-terminusofthegrowingproteinchain.

MultiplereadingframesinanmRNAsequence.

Howmanyframesarepossible?

MultiplereadingframesinanmRNAsequence.

KnownDeviationsfromtheUniversalGeneticCode• Universalcodeestablishedearlyinevolution.• Minordifferenceswerelaterevolutionarydevelopments.

tRNA

5-20StructureoftRNAs.• (a)AlltRNA structures:

• foldintofourbase-pairedstemsandthreeloops• haveaCCAsequenceatthe3ʹend(acceptorstem),towhichanaminoacidisattachedbyanaminoacyltRNA transferase

• haveananticodontripletatthetipoftheanticodonloop• havepost-transcriptionallymodifiedA,C,G,andUresidues

• (b)3DmodelofthegeneralizedL-shapedbackboneofalltRNAs

Howtogetthespecificity?

Translatingnucleicacidsequenceintoaminoacidsequence.

• Step1:• Anaminoacyl-tRNA synthetase couplesaspecific

aminoacidviaahigh-energyesterbond(makingtheaminoacidactivated)toeitherthe2ʹor3ʹhydroxyloftheterminaladenosineinthetRNA thathastheproperanticodon(cognatetRNA).

• Theenergyoftheesterbondsubsequentlydrivestheformationofthepeptidebondslinkingadjacentaminoacidsinagrowingpolypeptidechainintheribosome.

• Proofreadingbytheaminoacyl-tRNA synthetasemaintainsaverylowerror.

• Theanticodonthreenucleotidesarelocatedinaloopwheretheyareaccessibleforcodon-anticodonbasepairing.

• Step2:TheanticodonthreebasesequenceinthetRNAbase-pairswithacomplementarycodoninthemRNAspecifyingtheattachedaminoacid.

Enzyme!

Nonstandardbasepairingatthewobbleposition.

1.HowmanytRNAs areexistedforaminoacidcodon?

• Manycellshavefewerthanthe61differenttRNAsrequiredtoperfectlybasepaireachaminoacidcodon.

• I(inosine)inthewobblepositioncanpairwiththreedifferent butsynonymouscodons(C,A,Uincodonthirdbaseposition)bearingthesameaminoacid.

• GorUinthewobblepositioneachcanpairwithtwocodons.

• Ainthewobblepositionisrareinnature.

Sincemostorganismshavefewerthan45speciesoftRNA, sometRNA speciesmustpairwithmorethanonecodon.

FundamentalMolecularGeneticMechanisms

5.4StepwiseSynthesisofProteinsonRibosomes• Ribosomestructureandfunction• Translationmechanism– proteinsynthesis

Structureofthebacterialribosome.• Ribosome:proteinsynthesisorganelle/machine

• rRNA inribosome:thirdRNArequiredforproteinsynthesis(inadditiontomRNA,andtRNA)

• Ribosomesdifferinbacteria,archaea,andeukaryotes,butsharestructuralandfunctionalsimilarities.

• Ribosome(T.thermophilus)structure:

• small(30S)subunit– 16SrRNA andproteins

• large(50S)subunit– 23Sand5SrRNAs andproteins

• internalpositionsoftRNAs intheA,P,andEsites andelongatingpeptideattachedtothetRNA inthePsite.

The S is an abbreviation for "Svedberg" the unit of molecular size named after the Nobel prize winner who developed methods of determining molecular size by centrifugation.

RNAgelelectrophoresis

Reason?

1.Aminoacyl-tRNA binding, or A-site2.Peptidyl-tRNA binding, or P-site3.tRNA exit site, or E-site

RibosomeComponents• Ribosomessharestructuralandfunctionalsimilarities,butdifferinnumberofrRNAs andproteinsinbacteria,archaea,andeukaryotes.

• S(svedberg)unitsareameasureofthesedimentationrateofmacromoleculescentrifugedunderstandardconditions.

Comparisonofthecommoncorestructureatthecenterofribosomesfromalldomainsoflifeandbacterial,yeast,andhumanribosomes.• (a)RNAinthecommoncorestructure(lightblue)andproteindomainscommontoallribosomes(pink).

Initiationoftranslationineukaryotes.

Eightinitiationstepsinvolvingeukaryotictranslationinitiationfactors(eIFs)andGTPhydrolysis,whichstabilizessomecomplexes:

Chainelongationineukaryotes.• DuringchainelongationeachincomingtRNA movesthroughthreesites- A,P,andE.

Terminationoftranslationineukaryotes.• Translationisterminatedbyreleasefactorswhenastopcodon(UAA,UGA,UAG)isinAsite.

https://www.youtube.com/watch?v=5bLEDd-PSTQ

CircularstructureofmRNAincreasestranslationefficiency.• CircularmRNA,polysomes,andrapidribosomerecyclingincreasetheefficiencyoftranslation.

• Polysome:structurewithmultipleindividualribosomessimultaneouslytranslatingaeukaryoticmRNA

• (a)force-fieldelectronmicrograph• (b)modelofproteinsynthesisoncircularpolysomes andrecyclingofribosomalsubunits

FundamentalMolecularGeneticMechanisms

5.5DNAReplication• Semiconservativereplicationmechanism

Meselson-Stahlexperiment.• ExperimentshowedthatDNAreplicatesbyasemiconservativemechanism:

• E.colicellsweregrowninamediumcontaining“heavy/H”nitrogen(15N)ammoniumsaltsuntilallcellularDNAwaslabeled.

TwoHypothesis!!

TwoHypothesis!!

DNApolymerasesrequireaprimertoinitiatereplication.• DNAsynthesisalwaysproceedsinthe5ʹ→3ʹdirection,becausechaingrowthresultsfromformationofaphosphoester bondbetweenthe3ʹoxygenofagrowingstrandandtheαphosphateofadNTP.

• DNApolymerasesrequireashort,preexistingRNAorDNAprimer strand(withfree3’hydroxyl)thatisbase-pairedtothetemplatestrandtobeginsecondstrandgrowth.

Leading-strandandlagging-strandDNAsynthesis.• DuplexDNAisunwound,anddaughterstrandsareformedattheDNAreplicationfork.

• DNApolymeraseaddsnucleotidestoagrowingdaughterstrandinthe5ʹ→3ʹdirection.

• Leadingstrand:synthesizedcontinuouslyfromasingleRNAprimer(red)atits5ʹend.

Laggingstrand

• (1)AvirallargeT-antigenhelicasehexamerunwindstheparentalDNAstrands,beginningatareplicationorigin.• (2)DNApolymeraseε (Polε)extendstheleadingstranduptothereplicationfork.• (3)ThePCNA“slidingclamp”keepsPolε stablyassociatedwiththereplicationfork.• (4)Multiplecopies oftheheterotrimericproteinRPAbindthelaggingstrand.• (5)AprimaseRNApolymerase-DNApolymeraseα(Polα)complexsynthesizesprimersforlagging-strandsynthesis.• (6)APCNA–Polδ complexbindsthe3ʹendofeachprimerandextendstheprimertosynthesizemostofanOkazakifragment.

Thinkwhat’snecessaryforreplication!!!

(Proliferating cell nuclear antigen)

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

BidirectionalreplicationinSV40DNA.

• ReplicationisinitiatedbybindingtwolargeT-antigenhexamerichelicasestothesingleSV40originandformationoftwooppositelyorientedreplicationforks.

• EM:centersofvarioussizedreplication“bubbles”wereaconstantdistancefromeachendoftheEcoRI cutSV40DNA,consistentwithchaingrowthintwodirectionsfromacommonoriginlocatedatthecenterofabubble.

BidirectionalmechanismofDNAreplication.• EukaryoticchromosomalDNAmoleculescontainmultiplereplicationoriginsseparatedbytenstohundredsofkilobases.

FundamentalMolecularGeneticMechanisms

5.6DNARepairandRecombination• DNAsequencechanges– copyingerrorsandtheeffectsofvariousphysicalandchemicalagents.

• Threeexcisionrepairsystems• baseexcisionrepair• nonhomologousendjoining• homologousrecombination

• DefectsinDNArepairareassociatedwithcancers

ProofreadingbyDNApolymerase.

https://www.youtube.com/watch?v=6O0qD6KCOVE

FundamentalMolecularGeneticMechanisms

5.7Viruses:ParasitesoftheCellularGeneticSystem• Virusgenomeandstructure• Virusinvasionandreplicationinhostcells• Viruslifecycles– lyticandnonlytic

Bacteriophage

DNAgenome

• Step1:WhenviralcapsidproteinsatthetipofthetailinT4interactswithspecificreceptorproteinsontheexteriorofthehostcell(adsorption),theviralgenomeisinjectedintothehostcell(penetration).

• Step2:Host-cellenzymestranscribeviral“early”genesintomRNAs,whichhost-cellribosomestranslateintoviral“early”proteins.

• Step3:TheearlyproteinsreplicatetheviralDNA(replication)andinduceexpressionofviral“late”proteins,includingcapsidandassemblyproteinsandenzymesthatdegradethehost-cellDNA,supplyingnucleotidesforsynthesisofmoreviralDNA.

• Step4:Progenyvirions assembleinthecell(assembly).

• Step5:Viralproteinslysethecell(release),andnewlyliberatedvirusesinitiateanothercycleofinfectioninotherhostcells.

Lyticreplicationcycleofanenvelopedanimalvirus.

What’sdifference?

Releaseofprogenyvirions bybudding

Retrovirallifecycle.

• Step1:Viralenvelopeglycoproteinsinteractwithaspecifichost-cellmembraneprotein,causingtheenvelopetofusedirectlywiththeplasmamembrane,allowingentryofthenucleocapsid intothecytoplasmofthecell.

• Step2:ViralreversetranscriptasecopiestheviralssRNA genomeintoadouble-strandedDNA.

• Step3:TheviraldsDNAistransportedintothenucleusandintegratedintooneofmanypossiblesitesinthehost-cellchromosomalDNA.

• Step4:TheintegratedviralDNA(provirus)istranscribedbythehost-cellRNApolymerase,generatingviralgenomicRNAmolecules(brightred)andviralmRNAs(darkred),whicharetranslatedbyhost-cellribosomesintoglycoproteinsandnucleocapsidproteins.

• Step5:Progenyvirions assembleandarereleasedbybudding.

Virusdiseases

• Retrovirusesinfectonlycertaincelltypes• HTLV:leukemia• HIV-1:AIDS

• DNAvirusesintegrateintohost-cellgenome• HPVexpressionofoncogenes– cervicalcancer

Discussionwithfriends

• YousynthesizedDNAsequencesusingDNApolymerase,butgotseveralwrongsequences.Whichstepyouwillcheckfirst?

• Differentfromeukaryoticgenome,prokaryotehascirculargenome.Thecirculargenomehasoneprobleminitsreplicationprocess,especiallyattheendofreplication.What’stheproblemandhowdoesprokaryotesolvethis?

• Ifyouwanttousethevirusesforexogenousgeneexpressionincellline,whichprocessesshouldberemoved forthesafeandstablegeneexpression?

참고 (etc)