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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. www.BioInteractive.org January30,2017 Page1of5 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. www.BioInteractive.org January30,2017 Page2of5 Hands-on Activity HIV Reverse Transcription and AZT Student Handout 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! www.BioInteractive.org January30,2017 Page3of5 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. www.BioInteractive.org January30,2017 Page4of5 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. www.BioInteractive.org January30,2017 Page5of5