Download Student Handout Hands-on Activity HIV Reverse Transcription and

Hands-on Activity
HIV Reverse Transcription and AZT
Student Handout
HIVREVERSETRANSCRIPTIONANDAZT
INTRODUCTION
Thehumanimmunodeficiencyvirus(HIV)infectsanddestroyscellsoftheimmunesystem,weakeningaperson’sability
tofightotherinfectionsanddiseases.Acquiredimmunodeficiencysyndrome(AIDS)occurswhenaperson’simmune
systemisseverelycompromised.Withouttreatment,mostpeoplewithAIDSdie.Fortunately,researchershave
developeddrugstotreatHIVinfection.
Acombinationtherapy,ordrugcocktail,ofseveraldrugsatonetimemakesanHIVinfectionatreatablechronicdisease
ratherthanaterminalillness.Thedrugazidothymidine(AZT)isoneofthedrugscommonlyusedinthisdrugcocktail.
AZTtargetsacriticalstepintheHIVreplicationcycle:reversetranscription.
HIVhasasingle-strandedRNAgenome.DuringreversetranscriptionanHIVenzymeconvertstheHIVRNAintoDNAso
thatitcanbeinserted,orintegrated,intothehostcellgenome.Reversetranscriptionisdifferentfromusual
transcription,whereDNAistranscribedtoRNA.Inthisactivity,youwilllearnhowreversetranscriptionandAZTwork.
First,however,let’sreviewthestructuresofDNAandRNA.
DNAandRNAReview
Youprobablyknowthattheinstructionsforbuildingalltheproteinsthatmakeupananimalorplantcellarestoredin
DNA.DNAisanucleicacid.RNAisalsoanucleicacid.ThegenomeofHIVismadeofsingle-strandedRNA.
Nucleicacids(DNAandRNA)aremadeupofasequenceofnucleotides.Allnucleotideshaveacommonstructure:a
phosphategrouplinkedtoasugarmolecule(riboseinRNAanddeoxyriboseinDNA,Figure1)thatisinturnlinkedto
oneoffournitrogenousbases(Figure2).Thebasesadenine,guanine,andcytosinearefoundinbothDNAandRNA;
thymineisfoundonlyinDNA,anduracilisfoundonlyinRNA.ThebasesareoftenabbreviatedA,G,C,T,andU,
respectively.
Figure1.Thesugarsofnucleicacids.Allnucleicacidscontainafive-carbonsugarmolecule.InRNAthesugarmoleculeisriboseandinDNAitis
deoxyribose(thesecondcarbonhasahydroxylgroupandahydrogeninriboseandtwohydrogensindeoxyribose).Byconvention,thecarbon
atomsofthesugararenumberedwithprimes.
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Hands-on Activity
HIV Reverse Transcription and AZT
Student Handout
Figure2.Nucleosidesandnucleotides—what’sthedifference?Asugarmoleculeboundtoabaseiscalledanucleoside.Thenucleosidesare
thymidine,adenosine,guanosine,cytidine,anduridine.Whenthenucleosideisboundtoaphosphategroup,itiscalledanucleotide.
DuringRNAorDNAsynthesis,thehydroxylgrouponthe3ʹcarbonofthesugarofonenucleotideformsabondwiththe
phosphateofanothernucleotide.Inthisway,onenucleotideisjoinedtoanothertoformachainthatlengthensinthe5ʹ
(phosphate)to3ʹ(hydroxyl)direction(Figure3).
Figure3.TwoillustrationsofDNAnucleotidechains.TheleftsideofthefigureshowsthechemicalstructureofaDNAsequencethatisthree
nucleotideslong.The5’endhasafreephosphategroup(attachedtothe5’carbonoftheribose),andthe3’endhasafreehydroxyl(OH)group.
Whenanewnucleotideisattached,itsphosphategroupwouldformabondwiththe3’hydroxylgroup,sothesequencegrowsinthe5’to3’
direction.Therightsideofthefigureshowsasimplifiedwayofillustratingthesamesequence.Although,strictlyspeaking,thelettersA,G,C,T,and
Ustandforbases,theyarealsooftenusedindiagramstorepresentthewholenucleotidescontainingthesebases.Byconvention,DNAsequences
arewritteninthe5’to3’direction.
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Hands-on Activity
HIV Reverse Transcription and AZT
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AZTisstructurallyverysimilartothenucleosidethymidine(Figure4).Inacell,AZTcanbecomelinkedtoaphosphate
grouptoformanucleotidethatcanbeincorporatedinagrowingnucleicacidchain.
Figure4.AZTisstructurallysimilartothymidine.Thymidineisanaturallyoccurringnucleoside.AZTisadrug.
HIVcontainsanenzymecalledreversetranscriptasethattranscribesRNAintoDNA.WhenHIVinfectsacell,thereverse
transcriptasetranscribestheviralRNAgenomeintoacomplementaryDNAstrand.Theenzymethencatalyzesthe
synthesisofasecondDNAstrand,complementarytothefirst.Thedouble-strandedviralDNAistheninsertedintoahost
cell’schromosome.Thehostcell’sRNApolymerasetranscribestheviralDNAintoRNAmoleculesthatcanservebothto
provideinstructionsforthesynthesisofviralproteinsandasgenomesfornewviruses.
Let’sexaminethisprocess!
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Hands-on Activity
HIV Reverse Transcription and AZT
Student Handout
BACKGROUNDQUESTIONS
1.Drawasequenceof10RNAnucleotidestorepresentasegmentoftheHIVgenome.Youcanjustusetheshorthand
versionwithastringofletters.MakesureyouuseeachofthefourRNAnucleotidesatleastonce.
2.ThefirststepinreversetranscriptionisthesynthesisofacomplementaryDNAstrand.DrawadiagramoftheRNA
segmentfromstep1pairedwithDNAnucleotidebases.
3.ThesecondstepisthesynthesisofaDNAstrandcomplementarytothefirsttoproducedouble-strandedviralDNA.
Drawadiagramofadouble-strandedDNAmolecule.
4.CompareyourdrawingoftheRNA/DNAnucleotidebasepairs(inQuestion2)totheDNAnucleotidebasepairs(in
Question3),andineachdrawing,circlethenucleotidebasepairsthataredifferent.
5.Thedouble-strandedDNAisincorporatedintothehostcellDNA.WhatisthefunctionofDNAincells?
ACTIVITY
YourteacherwillprovideanactualsequenceofHIVRNA(writtenasaseriesofletters)andasetofDNAnucleotides.
Usingthenucleotides,synthesizeaDNAstrandcomplementarytotheviralRNAtemplate.Thenucleotidesshouldbe
assembledasfollows(Figure5).
Figure5.ExampleofhowthegrowingDNAchainisassembled.Eachnucleotidecutouthasatabthatsticksoutwhichrepresentsthe5’phosphate
groupthatattachestoastickysquarethatrepresentsthe3’OHgroup.
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Hands-on Activity
HIV Reverse Transcription and AZT
Student Handout
6.RecordthesequenceofthenewlysynthesizedDNAstrandbelowandthentakeapartyourDNAsequence.
7.NowreplaceoneofyourTnucleotideswithAZT.AZTissimilarinmolecularstructuretothymidinebutwithone
importantdifference.ExamineFigure3showingthechemicalstructuresofthymidineandofAZT.Whatarethe
differencesbetweenthetwostructures?
8.IncellswhereAZTispresent,AZTcanbeincorporatedinagrowingDNAchaininplaceofathymidinebase.Predict
whatmighthappenifAZTisincorporated.
9.Repeatstep6aboveinacellwhereAZTispresent.WritedownthesequenceoftheDNAyouconstructed.
10.Howisthissequencedifferentfromthesequenceinstep6?
11.Usingtheinformationabove,explainhowAZTaffectstheprocessofreversetranscription.
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