10-DNA & Gene Expression 6e Gene...Bio 6 – DNA & Gene Expression ... the copying or...

Bio6–DNA&GeneExpressionLab

OverviewInthislaboratoryyouwillinvestigatethreeaspectsofgenes:1)thestructureofDNA-themoleculeofwhichgenesaremade;2)thecopyingorreplicationofDNAasitoccursduringSphaseofthecellcycle,and3)theexpressionofgenesintotheproteinproductstheyencode.Inaddition,youwilllearnhowtoisolateDNAfromcells

IntroductionDeoxyribonucleicacid,orDNAforshort,isanucleicacidcommonlyreferredtoas“geneticmaterial”.Asyou learned inaprevious laboratory,genesareactually specific segmentsofchromosomeswhicharesimplyreally longpiecesofDNA!SogiventheobviousimportanceofDNAwewanttomakesureyouunderstandDNAstructure,howDNAiscopiedincells,andhowindividualgenesareexpressedinto“geneproducts”,whichformostgenesisaspecificprotein.

Part1:DNAIsolationDNAissurprisinglyeasytopurifyfromplantandanimaltissues.Youmayrecallfromanearlierlabthatchromosomes, which are made of DNA, are stored in the nuclei of eukaryotic cells. So to purifychromosomalDNAyouneedtosomehowreleaseitfromthecellnucleiandthenseparateitfromallothercellularmaterials.Todothiswillrequirethreegeneralsteps:

1) breakingopencellsinthetissuetoreleasetheDNAfromcellnuclei

• thisisdonebymashingthetissueinasolutionwithdetergentandsalttobreakupcellmembranes

2) removingtheinsolublematerial(theDNAremainsdissolvedinliquidsinceitissoluble)

• thisisdonebyfilteringthetissue“mash”andtoisolatetheliquidportion

3) precipitatingtheDNAintheliquidcollected(makingtheDNAinsoluble)

• thisisdonebyaddingtherightamountsofsaltandalcoholtomaketheDNAinsolubleNowthatyouhaveabasicunderstandingoftheprinciplesinvolved,let’spurifysomeDNAfromstrawberrytissue…

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Exercise1–PurificationofDNAfromstrawberrytissueObtainyourmaterialsfromthefrontofthelab,carefullyfollowthestepsbelow,andinabout5minutesyouwillhaveisolatedstrawberryDNA!

1. Add4mlofdetergent/saltsolutiontothestrawberrysliceinaZiplocbag.

2. RemovemostoftheairfromtheZiplocbag,sealit,andthoroughlymashthestrawberrysliceinthedetergent/saltsolution(squeezewithhandsorpressontabletop).

3. Setupafunneloveraglassbeakerandlinethefunnelwith2ormorelayersofcheesecloth(foldthecheeseclothinto2ormorelayers).

4. TransferasmuchofthestrawberrymashasyoucanfromtheZiplocbagtothecheeseclothinthefunnel(ifyoulikeyoucancutacornerofthebagandsqueezeoutitscontents).

5. Allowtheliquidfromthestrawberrymashtopassthroughthecheeseclothandcollectinthebeaker(youcansqueezethecheeseclothtohelpasmuchliquidaspossiblepassthrough).

6. Add10mloficecoldethanoltotheliquidcollectedinthebeakerandmixthoroughlybyswirlingthebeaker.

7. Usedahookedglassrodtostir&collecttheprecipitatedDNA.

Part2:DNASTRUCTURE&DNAREPLICATION

DNAStructureLiketheproteinsandpolysaccharidesyoulearnedaboutearlierinthecourse,DNAisapolymer.Recallthatapolymerisachainofsmallermoleculesormonomers.Forexample,proteinsarepolymersofaminoacids(i.e.,aminoacidsarethemonomersinproteins).SoifDNAisapolymerofsmallermonomers,whatarethemonomersinDNA?Theanswerisnucleotides.

All nucleotides in DNA have the same basicstructure:thesugardeoxyriboseconnectedtoaphosphategroupononesideandtooneoffourpossible nitrogenous bases (“bases” for short)ontheother.Thephosphategroupisacidicandthusnegativelycharged.ThisiswhyDNAhasanetnegative charge.Becauseall nucleotides inDNA contain deoxyribose they are calleddeoxyribonucleotides, thoughforsimplicitywewill just call them “nucleotides”. As shownbelow,thefourdifferentnucleotidesinDNAareeach referred to by their base: adenine (A),cytosine(C),guanine(G)andthymine(T).

phosphate group

sugar (deoxyribose)

nitrogenous base

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ThesefournucleotidesshownabovecanbelinkedtogetherinanyordertoformanucleotidepolymerthatisreferredtoasasinglestrandofDNAliketheexampleshownbelow.

Noticethateachnucleotideisconnectedtothenextviathesugar(deoxyribose)andphosphate,thus forming what is called the “sugar-phosphatebackbone”ofaDNAstrand.Noticealso that each end of the sugar-phosphatebackbone isdifferent: whatwecall the5’endhasafreephosphategroup,whiletheotherendhas a free hydroxyl (–OH) group on the sugar,whatwecallthe3’end.The5’to3’orderofthebases in each nucleotide of a DNA strandconstitutesits“DNAsequence”.ThebaseofeachnucleotideinastrandofDNAsticksoutperpendiculartothesugar-phosphatebackbone. This is important because DNA isactually adouble-strandedmolecule. The twostrandsofaDNAmoleculeareheldtogetherbyinteractionsbetweenthebasesoneachstrand,aphenomenonknownasbasepairing.

Onthenextpagearethreedifferentillustrations,eachrepresentingadouble-strandedDNAmolecule.Noticethebasepairingbetweenthetwostrandsandthattheyareanti-parallel(the5’and3’endsare

OH 3’

5’

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orientedintheoppositedirection).Thetwostrandsalsoformacoilorhelix,hencethereferencetoDNAasadouble-strandedhelixorsimplya“doublehelix”.

Thebase-pairinginaDNAmoleculeinvolveschemicalattractionsbetweencertainbasesduetohydrogenbonding.Forbasepairingtooccur,thebasesmusthavecomplementarychemicalgroups(e.g.,partialnegativematchedwithpartialpositiveorviceversa)andcomplementarysizes(onelargerpurine–AorG–andonesmallerpyrimidine–CorT).Forthesetworeasons,thebasepairinginDNAislimitedtoadeninewiththymine(A:Tbasepairs)andguaninewithcytosine(G:Cbasepairs):

ThefactthateachbasecanpairwithonlyonepartnergivesDNAaremarkableproperty:thesequenceofoneDNAstranddeterminesthesequenceoftheother.Forexample,ifthesequenceofoneDNAstrandisallA’s,theotherstrandmustbeallT’sforbasepairingtooccurbetweenthestrands.Let’sconsideranotherexamplestartingwiththesequenceofonestrand:

5’ – C A T G C A A C G T C C A A A T T A G T – 3’

G C

T A

A T

G

G

C

CA T

GC

T A

T A

A T

A T

G CA T

O

O

OH–O

P

O O–O P

O

OO

P–O

–O OP

OO

O

OH

H2C

H2C

H2C

H2C

O

O

O

O

O

O

O

O

PO–

O–

O–

O–

OH

HO

O

O

O

P

P

P

O

O

O

O

O

O

O

O

T A

G C

C G

A TCH2

CH2

CH2

CH2

5’

5’3’

3’

d+

d+d-

d-d- d+

d+

d+

d-

d-

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SinceeachstrandofDNAmoleculemustbecomplementary,thetwostrandsmustbeinananti-parallelorientation and have nucleotides thatwillbase pair withall the nucleotides on the opposite strand.Therefore,thedoublestrandedsequenceofthisshortDNAmoleculemustbe:

5’ – C A T G C A A C G T C C A A A T T A G T – 3’ 3’ – G T A C G T T G C A G G T T T A A T C A – 5’

Lookcarefullyatthissequenceandyouwillseethateachstrandis,infact,anti-paralleltotheother,andallofthenucleotidespositionedacrossfromeachothercanformabasepair(A:TorG:C). Thesetwocriteriaareessentialforthestrandstointeractwitheachotherviabasepairingtoformadoublehelix.SoifyouknowthesequenceofoneDNAstrandyoucaneasilydeterminethecomplementarysequenceoftheotherstrandsincethereisonlyonepossibility.AtthispointyouknowenoughaboutDNAstructuretodothenextexerciseinwhichyouwillbuildamodelofdouble-strandedDNAafterbeinggiventhesequenceofasingleDNAstrand…Exercise2A–Buildingadouble-strandedDNAmoleculeTobuildyourDNAmoleculeusethemodelkitandkeytothepartsatyourtable.Yourinstructorwillassignyourgroupasingle-strandDNAsequenceafterwhichyouwillfollowtheinstructionsbelow:

1. Onyourworksheet,writethesingle-strandDNAsequenceyou’vebeengiven,indicatingthe5’and3’ends,andrefertothehandouttofamiliarizeyourselfwiththecomponentsofyourDNAmodelkit.

2. UsetheDNAmodelkittobuildeachnucleotideinyoursinglestrandsequenceseparately.

3. Assemblethenucleotidesintheorderofyoursequence,beginningatthe5’end.

4. Writethecomplementarysequencetoyouroriginalsingle-strandsequenceonyourworksheet.

5. Buildthecomplementarysequenceasyoudidinsteps2and3above.

6. PutthetwoDNAstrandsthatyou’vejustbuilttogethertoformadouble-strandedDNAmolecule,beingsuretheyareanti-parallelandthatthebasepairsmatchupproperly.

NOTE:SavetheDNAmoleculeyouhavejustbuiltforuseinExercise2B.

DNAReplicationInapreviouslabyoulearnedthatwhenacellentersthecellcycle,inordertodivideitmustfirstcopyallofitschromosomes–allofitsDNA.This,asyoushouldrecall,willoccurduringSphaseofthecellcycle(SforDNASynthesis).TheprocessofcopyingDNA,DNAsynthesis,isformallyknownasDNAreplication.DNAreplicationisanextremelycomplexprocessinvolvingmanydifferentenzymesplayingspecificrolesintheprocess.Inthelecturepartofthiscourseyouwilllearnaboutsomeoftheseenzymes,howeverforthepurposeofthislaboratorywewillnotbeconcernedwiththesedetails.Wesimplywantyoutolearnhowtheprocessunfolds,knowingthatmanydifferentenzymesarerequired.

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The process of DNA replication depends on base pairing between nucleotides. As it turns out, eachoriginalDNAstrandisusedasatemplateorguidetoproduceacomplementaryDNAstrand.Thisrequiresthatthebasepairsbetweenthetwooriginalstrandsbedisrupted,thus“unzipping”theDNA. Anewstrandwithcomplementarynucleotidesisthenproducedforeachoriginalstrand.ThenetresultistwoidenticalcopiesoftheoriginalDNAmolecule!Let’srevisittheDNAsequenceintheexamplefromtheprevioussectiontoseehowthiswouldwork:

5’ – C A T G C A A C G T C C A A A T T A G T – 3’ 3’ – G T A C G T T G C A G G T T T A A T C A – 5’

FortheaboveDNAmoleculetobecopiedbyDNAreplication,thetwostrandsmustfirstbeseparated:

5’ – C A T G C A A C G T C C A A A T T A G T – 3’

3’ – G T A C G T T G C A G G T T T A A T C A – 5’

Onceseparated,eachstrandcanbeusedasatemplatetoproduceacomplementarystrand.Eachnewcomplementarystrandmust,ofcourse,beanti-parallel,andtheenzymesthatsynthesizethenewstrandscanonlydosoina5’to3’directionasindicatedbelow:

5’ – C A T G C A A C G T C C A A A T T A G T – 3’ 3’ - G G T T T A A T C A – 5’

5’ – C A T G C A A – 3’ 3’ – G T A C G T T G C A G G T T T A A T C A – 5’

Eachnewcomplementarynucleotidecanonlybeaddedtothe3’endofthegrowingnewstrand,onenucleotideatatime.Oncetheprocessiscomplete,youcanseethattheoriginalDNAmoleculehasbeenaccuratelyreplicatedina5’to3’direction:

5’ – C A T G C A A C G T C C A A A T T A G T – 3’ 3’ – G T A C G T T G C A G G T T T A A T C A – 5’

5’ – C A T G C A A C G T C C A A A T T A G T – 3’ 3’ – G T A C G T T G C A G G T T T A A T C A – 5’

NowthatyouhaveabasicunderstandingofDNAreplicationyouarereadytocompletethenextexercise…

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Exercise2B–ReplicationofaDNAmoleculeReplicatetheDNAmoleculeyoubuiltinExercise2Abyfollowingtheinstructionsbelow.Althoughyoumayintuitivelysee how it all fits together, please DO NOT deviate from the instructions. It is important that you follow theinstructions exactly to reinforcehow this processworks in cells. This is thewhole point of the exercise! You’rebasicallyplayingtheroleofDNApolymerasewhichcanonlyreplicateDNA,withthehelpofseveralotherenzymes,inthemanneroutlinedbelow:

1. Completely“unzip”yourDNAmoleculebyseparatingthetwostrands.

2. Workingwithonestrandatatime,identifythenucleotideatthe3’endofthemolecule.

3. Buildanucleotidecomplementarytothisnucleotideandbasepairthenewnucleotidewiththenucleotideatthe3’endoftheoriginaltemplatestrand.

4. Buildanucleotidecomplementarytothenextnucleotideinthetemplatestrandandaddittothe3’endoftheprecedingnucleotide,basepairingitwiththetemplatestrand.

5. Repeatstep4untilthecomplementarystrandiscomplete.

6. Repeatsteps2through5withtheotheroriginalstrandtocompletetheDNAreplicationprocess.

NOTE:SavetheDNAmoleculesyouhavejustproducedforuseinExercise3A.

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Part3:GENEEXPRESSION

Agene ismuch likea recipe inacookbook,withachromosomebeing like thecookbook itself. Geneexpressioninthisanalogywouldbeequivalenttousingarecipeinthecookbooktomakeafooditem.However instead of instructing how to prepare a food item, a gene contains instructions on how toconstructaprotein(orinsomecasesanRNAmolecule).LikeDNAreplication,geneexpressionisverycomplicatedandinvolvesmanydifferentenzymes.Thus,wewillleavemostofthesedetailstothelectureportionofthecourseandsimplyaddresshowtheprocessunfolds in general. The process of gene expression requires two distinct cellular processes: 1) thetranscriptionofDNAencodingtheproteinintoaverysimilarnucleicacidpolymercalledRNA(ribonucleicacid),and2)thetranslationoftheRNAsequenceintoaprotein–apolymerofaminoacids.HowaDNAsequence encodes the amino acids in a protein involves the “genetic code”, something we will alsoaddress.Beforewelookintotheseconcepts,however,wefirstneedtobecomefamiliarwithRNA.

RNARNA,likeDNA,isapolymerofnucleotides.ThenucleotidesfromwhichRNAismade,however,containthesugarribosewhichhasonemore–OHgroupthandeoxyribose.ThenucleotidesinRNAarethuscalledribonucleotides, though for simplicity wemay also refer to them as “nucleotides” as we have doneregardingDNA.Thebasesinribonucleotidesarethesameasindeoxyribonucleotideswithoneexception:thebaseuracil(U)isusedinplaceofthymine(T).Belowisadiagramillustratingthesedifferences:

OneotherkeydifferencebetweenRNAandDNAisthatRNAexistsasasingle-strandedmolecule.EventhoughRNAissingle-stranded,itsnucleotidesstillparticipateinbase-pairingasyouwillsee,withuracil(U)formingbasepairswithadenine(A).

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TranscriptionReturningtothecookbookanalogy,transcriptionisessentiallymakingaphotocopyofarecipe.TherecipeistheDNAsequenceencodingtheinstructionstobuildaprotein,andRNAservesasasimplephotocopyoftheoriginalrecipe.Thisisaprettygoodanalogy,howeveritdoesnottakeintoaccountthetwostrandsofaDNAmolecule,eachhavingadifferentsequence. Infact,onlyoneDNAstrandofageneactuallycontainstheinstructionsforbuildingaprotein,thestrandwecallthecodingstrand.Thecomplementarystrand,calledthetemplatestrand,doesnotcontainanyinstructionsyetitisextremelyimportantintheprocessoftranscription.TranscriptionisactuallyverysimilartoDNAreplicationinthatDNAis“unzipped”andusedasatemplatetomakeacomplementarystrandofRNAinsteadofDNA.UnlikeDNAreplication,thisoccursonlywithinasinglegeneatatime(DNAreplicationresultsinthecopyingofentirechromosomes),andonlyoneDNAstrand, the template strand, is used tomake RNA. The resulting RNA is complementary to theDNAtemplatestrandandthusacopyoftheDNAcodingstrandsequence,withuracil(U)inplaceofthymine(T).Toillustratethis,let’spretendtheDNAsequenceweusedearlieristobetranscribed:5’ – C A T G C A A C G T C C A A A T T A G T – 3’coding 3’ – G T A C G T T G C A G G T T T A A T C A – 5’template 5’ – C A T G C A A C G T C C A A A T T A G T – 3’coding

5’ – C A U G C A A – 3’ 3’ – G T A C G T T G C A G G T T T A A T C A – 5’template 5’ – C A T G C A A C G T C C A A A T T A G T – 3’coding 3’ – G T A C G T T G C A G G T T T A A T C A – 5’template

5’ – C A U G C A A C G U C C A A A U U A G U – 3’RNA

Asyoucansee,theRNAproducediscomplementarytothetemplatestrandandidenticalinsequencetothecodingstrand,withU’sinplaceofT’s.Inourcookbookanalogy,wehavejustcreatedaphotocopyoftherecipe!TheRNAmoleculeisnowreadytobeusedintheprocessoftranslationtomaketheproteinencodedbyitssequence.Itisimportanttorealizethat,eventhoughallRNAmoleculesaremadebytheprocessoftranscription,theycanbeusedinavarietyofways.AtthemomentweareonlyconsideringoneroleforRNA,servingasacopyofthecodingstrandinagene.RNAusedforthispurposeiscalledmessengerRNAormRNAforshort.AnotherroleforRNAwillbeaddressedwhenwelookattheprocessoftranslation.

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Let’smoveontothenextexercisereinforcingtheconceptoftranscription…Exercise3A–TranscriptionofDNAintoRNAYourgroupwilltranscribetheDNAmoleculesyouproducedinExercise2B.Todosoyouwillconsiderthestrandwiththeoriginalsequencegivenbyyourinstructorasthecodingstrand,andtheotherstrandasthetemplatestrand.Onceyouhaveidentifiedyourcodingandtemplatestrands,proceedasdescribedbelowkeepinginmindthatthisishowtranscriptionoccursincells:

1. Completely“unzip”yourDNAmoleculebyseparatingthetwostrands.

2. Identifythenucleotideatthe3’endofthetemplatestrand.

3. Buildaribonucleotidecomplementarytothisnucleotideandbasepairitwiththenucleotideatthe3’endoftheDNAtemplatestrand.

4. Buildaribonucleotidecomplementarytothenextnucleotideinthetemplatestrandandaddittothe3’endoftheprecedingribonucleotide,basepairingitwiththeDNAtemplatestrand.

5. Repeatstep4untilthecomplementarymRNAiscomplete.

6. UnzipthemRNAfromtheDNAtemplatestrand,andrestoretheDNAtoitsoriginaldouble-strandedstate.

NOTE:SavetheRNAmoleculeyouhavejustproducedforuseinExercise3C.

TheGeneticCodeBeforewelookattranslation,youneedtounderstandthegeneticcode.Whilethismayseemlikeyetanother thing you need to learn in biology class, having knowledge of the genetic code is truly aremarkableprivilege.Scientistsandthinkersfromthepastwouldhavegivenanythingtoknowwhatyouare about to learn, arguably the most fundamental biological process there is: how genes storeinformation,informationintheformofaDNAsequencethatcanbeexpressedintoproteinsandpassedon to thenext generation. Inotherwords, youare about to learnwhat genetic informationactuallymeans,notjustforhumanbeings,butforalllifeonearth.OnceitwasknownthatgenesaremadeofDNAwhichsomehowcodesforproteins,anumberofscientistssetouttouncovertheunderlyinggeneticcode.Sinceproteinsarepolymersofaminoacids,theyreasonedcorrectly that the sequence of nucleotides in DNA (ormore specifically the sequence of bases)mustsomehowencodespecificaminoacidsandtheirorderinaprotein.RecallfromLab4thatthereare20aminoacidsusedtomakeproteins.Thisfactwaswellknownintheearly1960swhentheseinvestigationsbegan,thusthegeneticcodewasassumedtoconsistofatleast20differentshortnucleotidesequences,presumablyofthesamelength.Giventhatthereareonly4differentonenucleotidesequences(A,C,G,T),16differenttwonucleotidesequences(AA,AC,AG,AT...),and64differentthreenucleotidesequences(AAA,AAC,AAG,AAT…),acodeconsisting of three nucleotide sequences was clearly the best candidate. Through some very cleverexperimentationthegeneticcodewassoondiscoveredtobejustthat,a3-nucleotidecodewhichisreadina5’to3’direction.Wenowrefertoeach3-nucleotidecombinationinthegeneticcodeasacodon.Themeaningsofall64codonshavebeendeterminedandarerepresentedinthefollowingchart:

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Thischartusesuracil(U)insteadofthymine(T)sinceRNA,notDNA,isusedtomakeproteinsduringtheprocessoftranslation(thoughbothnucleotideshavethesamemeaninginthegeneticcode).Thechartisorganizedtomakeiteasytofindanyparticularcodon.Codonsineachrowbeginwiththesamebase,codonsineachcolumnhavethesamesecondbase,andcodonsineachboxdifferonlyinthethirdbase.Ifyoulookcarefullyyouwillnoticethatall20aminoacidsarerepresentedinthiscode,withsomebeingrepresentedbyonlyonecodon(e.g.,UGGfortryptophan),andothersbeingrepresentedbyasmanyassixcodons(e.g.,leucine).Youwillalsonoticethatthereisasinglestartcodon(AUG,whichalsocodesformethionine), aswell as three stop codons (UAA,UAG,UGA). The importanceof these codons in theproduction of a polypeptide will be addressed as wemove on to the final step of gene expression,translation,butnotbeforeyoucompletethenextexercise.

Exercise3B–UnderstandingthegeneticcodeUsethechartofthegeneticcodeonthispagetocompletethecorrespondingexercisesonyourworksheet.

Translation

AmessengerRNAmolecule(mRNA)producedbytranscriptioninthenucleusofacellistransferredtothecell cytoplasmwhere its sequence is translated into proteins by ribosomes. This process, known astranslationorproteinsynthesis,also involvesanotherfunctionaltypeofRNA,transferRNA (tRNA forshort).EachtRNAisattachedoneofthe20differentaminoacids,readytodeliverittoaribosomewhenneeded,andalsocontainsa3-nucleotideanticodon.Theroleofribosomesisto:1)facilitatebase-pairingofmRNAcodonswithtRNAanticodons,and2)catalyzetheformationofpeptidebondsbetweenaminoacidsdeliveredbyconsecutivetRNAs.Thisbasicprocessisillustratedinthediagrambelow:

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Theprocessbeginswhenaribosomelocatesthestartcodon–AUG.Thisisextremelyimportanttoensurethattheribosomenotonlybeginswhereitissupposedto,butalsousesthecorrectreadingframe.Fromthispointtheribosome,workingwithtwocodonsandtwotRNAsatatime,willmoveina5’to3’directiondownthemRNAuntilitreachesastopcodonandendstheprocess.Toseehowimportantstartcodonsandreadingframesare,let’sconsiderthemRNAsequenceyouproducedearlier:

5’ – C A U G C A A C G U C C A A A U U A G U – 3 BeginningwiththeAUGstartcodonandmovinginthe5’to3’direction,aribosomewouldtranslatethismRNAsequenceintothefollowingpolypeptide(seeyourgeneticcodechart):

met–gln–arg–pro–asn(stop)

IftranslationwastobeginatCAUinsteadofthestartcodonAUG,theribosomewouldbeusingthewrongreadingframeandwouldproduceanentirelydifferentpolypeptide:

5’ – C A U G C A A C G U C C A A A U U A G U – 3

his–ala–thr–ser–lys–leuThereisalsoathirdreadingframewhichwouldyieldacompletelydifferentpolypeptideaswell:

5’ – C A U G C A A C G U C C A A A U U A G U – 3

cys–asn–val–gln–ile–serAsyoucansee,beginningtranslationatthestartcodonensuresthatthecodonsarereadcorrectlyandthe polypeptide is made correctly. In this way, a DNA sequence transcribed into an identical RNAsequencecanbeusedtoconsistentlyproducemanycopiesofthesamepolypeptide.

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Now thatyouunderstand theprocess, let’sputgeneexpression intocontext. Pretendyou justateastarchymeal,sayamashedpotato.Asaconsequence,yourbloodsugar(glucose)willgoupandspecialcellsinyourpancreaswillproducemoreinsulintohelprestoreyourbloodsugartonormallevels.Insulinisaproteinconsistingofasinglepolypeptide.Tomakemoreinsulin,thesepancreaticcellswillhavetoexpress the insulingene. Thismeans transcribing the insulingene intomRNAfollowedby ribosomestranslatinginsulinmRNAintoinsulinpolypeptides.Asingleinsulingene(youactuallyhavetwo,onefrommomandonefromdad)canyieldmanymRNAcopiesbytranscription,andeachmRNAcanyieldmanyinsulinpolypeptides.Thushugeamountsofthegeneproduct,inthiscasetheproteinhormoneinsulin,canbeexpressedfromasinglegene.

Exercise3C–TranslationofmRNAintoapolypeptideInthisexerciseyouwillfunctionasaribosomeandtranslatethemRNAmoleculesyouproducedinExercise3Aintoashortpolypeptide.Beforeyoudoso,besurethatallyourtRNAsareconnectedtothecorrectaminoacidandthenproceedexactlyasdescribedbelow:

1. IdentifytheAUGstartcodoninyourmRNAsequence.

2. CreateatRNAusingyourmodelkitthathasananticodoncomplementarytoAUGandanattachedaminoacid.

3. BasepairtheanticodonofthistRNAwiththeAUGstartcodoninthemRNA.

4. CreateatRNAusingyourmodelkitthathasananticodoncomplementarytothenextcodoninyourmRNAmoleculeandanattachedaminoacid(besureyouaregoingina5’to3’direction).

5. BasepairtheanticodonofthistRNAwiththemRNAcodon.

6. FormapeptidebondbetweentheaminoacidsoftheadjacenttRNAsandremovethefirsttRNA(thedipeptidejustformedshouldnowbeattachedonlytothesecondtRNA).

7. Repeatsteps4through6basedonthe3rdcodoninyourmRNAafterwhichyoushouldreleaseyourpolypeptidefromthelasttRNAwhichshouldthenbedetachedfromthemRNA.

NOTE:Althoughnotshowninthisexercise,inacellaribosomewouldfacilitatethisprocess.

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DNA&GeneExpressionLabWorksheet Name________________________ Ex.1–PurificationofDNAfromstrawberrytissue

Ø HowdidyoureleasetheDNAfromcellsinthestrawberryslice?Ø WhatdidyouaddtotheliquidfiltratetomakethestrawberryDNAinsoluble?Ø DescribetheappearanceofyourstrawberryDNAafteritwasprecipitated.Ø WhatpropertyofDNAwouldmakeitwatersoluble?DidanyofyourprecipitatedDNAappearto

dissolveinwater?Ex.2A–Buildingadouble-strandedDNAmolecule

Ø WritetheDNAsequenceyourgroupwasgivenbelow,thenwritethecomplementarystrandjustbelowit(besuretoalsoindicatethe5’and3’endsofeachDNAstrand).

Ø WhatdoesitmeantosaythatthestrandsinaDNAmoleculeareanti-parallel?Ø Whatchemicalgroupsidentifythe3’and5’endsofaDNAstrand?Ex.2B–ReplicationofaDNAmolecule

Ø Usingdifferentcolorsfor“old”and“new”DNAstrands,writetheDNAsequencesofbothDNAmoleculesresultingfromyourcompletionoftheDNAreplicationexercise.

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Ø BasedonyourmodelofDNAreplication,doestheoriginalDNAmoleculestayintact?Ø WhatismeantbythetermDNAtemplate?Ex.3A–TranscriptionofDNAintoRNA

Ø UsingdifferentcolorsforRNAandDNAstrands,writetheDNAsequenceofyouroriginalDNAmoleculeaswellastheRNAmoleculeproducedbytranscription.Asalways,besuretolabelthe5’and3’endsofeachstrand,andcorrectlylabeltheDNAtemplateandcodingstrands.

Ø DNAandRNAdifferinthreebasicways.Whatarethesethreedifferences?

Ex.3B–Understandingthegeneticcode

Ø Indicateallcodonsthatspecifytheaminoacidserine.Ø Ifthegeneticcodeconsistedofcodonswith4nucleotides,howmanydifferentcodonswouldthere

be?Ø DeterminetheaminoacidsequenceencodedbythefollowingmRNAsequence,anddon’tforgetto

beginwiththestartcodon:

5’–GCGUAUGACCGUUAUAGAUGGGCGUCUUCCACACUGAAUACUAACGAAU–3’

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Ø WhatisthereadingframeofaDNAsequence?Whyisthissoimportant?Ex.3C–TranslationofanmRNAintoapolypeptide

Ø WriteoutthemRNAsequenceyougeneratedbytranscriptioninExercise3A,andbelowthesequenceindicatetheaminoacidsequenceyourmRNAencodes:

Ø Theaminoacidsusedduringtranslationareattachedtowhatkindofmolecule?

Ø Describethetwobasicrolesofribosomesduringtheprocessoftranslation.

QuestionsforReview:

a) BrieflydescribetherolesofthefollowingenzymesinDNAreplication(inthebacteriumE.coli):

DNApolymeraseI DNApolymeraseIII

Helicase Topoisomerase(Gyrase)

Primase DNAligase

b) DescribetheroleofRNApolymeraseintranscription.

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