BIOSYNTHESISOFDNAANDRNA:REPLICATIONANDTRANSCRIPTIONHLeeYuJsuicoJunsay

DepartmentofChemistry

SchoolofScienceandEngineering

AteneodeManilaUniversity1

NucleicAcidsareimportantfortheirrolesinthestorage,transferandexpressionofgeneCcinformaCon.

2

HOW DO YOU TRANSFER INFORMATION TO THE NEXT GENERATION?

HOW DO YOU DECODE THE INFORMATION AND MAINTAIN THE CELL’S STRUCTURE AND FUNCTION?

TransferandinterpretaConofgeneCcinformaConisdescribedinthecentraldogmaofmolecularbiology.

3

REPLICATIONOFDNA

4

DNAispassedonthrureplicaConandissemiconservaCve.

5

DNAispassedonthrureplicaConandissemiconservaCve.

6

DNAispassedonthrureplicaConandissemiconservaCve.

7

DNAispassedonthrureplicaConandissemiconservaCve.

8

DNAispassedonthrureplicaConandissemiconservaCve.

9

ReplicaConstartatdiscretepointsonDNAandisbidirecConal

10

ReplicaConstartatdiscretepointsonDNAandisbidirecConal

11

ReplicaConstartatdiscretepointsonDNAandisbidirecConal

12

ReplicaConstartatdiscretepointsonDNAandisbidirecConal

13DNA_bi_direcConal

ReplicaConissemi‐disconCnuous.

14

LeadingstrandiscopiedconCnuously

Laggingstrandiscopiedinsegmentswhichmustbejoined

ReplicaConissemi‐disconCnuous.

15DNA_leading_lagging

16

•  IfWatsonandCrickwereright,thenthereshouldbeanenzymethatmakesDNAcopiesfromaDNAtemplate

•  In1957,ArthurKornbergandcolleaguesdemonstratedtheexistenceofaDNApolymerase‐DNApolymeraseI(PolI)

•  PolIneedsallfourdeoxynucleoCdes,atemplateandaprimer‐ass‐DNA(withafree3'‐OH)thatpairswiththetemplatetoformashortdouble‐strandedregion

17

DNAelongaConhappensthroughtheacConofDNAPolymerases.

 template‐directedenzymes 5classesareidenCfied allDNApolymerasehavecommonstructuralfeatures Klenowfragment

18

DNAelongaConhappensthroughtheacConofDNAPolymerases.

 specificitydictatedbyH‐bondingandshapecomplementaritybetweenbases

• bindingofcorrectbaseisfavorable(morestable)• interacConofresiduesintheenzymetotheminorgrooveofDNA• closedownaroundtheincomingNTP

19

DNAPolymerasecreatesthephosphodiesterbondfromapre‐exisCng3’‐OHandadNTPcreaCngapyrophosphate.

20

ThefirstDNApolymeraseswerediscoveredinE.coli.

21

TheexonucleaseacCvityofDNAPolymerasesareimportantforproofreading.

 proofreadingmechanisms• Klenowfragment–removesmismatchednucleoCdesfromthe3’’endofDNA(exonucleaseacCvity)• detecConofincorrectbase

‐ incorrectpairingwiththetemplate(weakH‐bonding)‐ unabletointeractwiththeminorgroove(enzymestalls)

22

TheexonucleaseacCvityofDNAPolymerasesareimportantforproofreading.

23

DNApolymeraseIIIholoenzymereplicatestheE.colichromosome.Severalotherproteinsarealsoimportant:

24

UnwindingofDNAhelix:

 dnaBhelicase–disruptsH‐bondsholdingthe2strands

 DNAgyrase–introducesnegaCvesupercoils

25

UnwindingofDNAhelix:SSB(single‐strandedDNA‐binding

protein)• bindstounwoundstrandstopreventannealing• formspreprimingcomplex

26

Primer

RNAprimesthesynthesisofDNA.

PrimasesynthesizesshortRNA.

27

ElongaConbyDNAPolymeraseIII

28

DNAPolymeraseIcleavesRNAprimerandinsertsdNTPs.

29

  DNAligasesealsbreaksinthedoublestrandedDNA  DNAligasesuseanenergysource(ATPineukaryotesandarchaea,NAD+inbacteria)toformaphosphodiesterbondbetweenthe3’hydroxylgroupattheendofoneDNAchainand5’‐phosphategroupattheendoftheother.

30

  DNAligasesealsbreaksinthedoublestrandedDNA  DNAligasesuseanenergysource(ATPineukaryotesandarchaea,NAD+inbacteria)toformaphosphodiesterbondbetweenthe3’hydroxylgroupattheendofoneDNAchainand5’‐phosphategroupattheendoftheother.

31

32

33

34

DNAreplicaConsites

35

DNAreplicaConsites

36

LinearDNAgetsshorteragereachreplicaConcycle.ToprotectDNA,TelomeresareaddedtotheendsofDNA.

DNA_telomeres

37

TelomeresaresynthesizedbyRibonuclearproteincomplexescalledTelomerases.

38

TelomerasesuseRNAtemplatetosynthesizeDNA.(REVERSETRANSCRIPTASE!)

  Primerrequired,butastrangeone‐atRNAmoleculethattheviruscapturesfromthehost

  RTtranscribestheRNAtemplateintoacomplementaryDNA(cDNA)toformaDNA:RNAhybrid

39

ReverseTranscriptaseisverycommoninviruses

  Three enzyme activities

 RNA-directed DNA polymerase

 RNase H activity - degrades RNA in the DNA:RNA hybrids

 DNA-directed DNA polymerase - which makes a DNA duplex after RNase H activity destroys the viral genome

  HIV therapy: AZT (or 3'-azido-2',3'- dideoxythymidine) specifically inhibits RT

RECAP:1. DNAreplicaConissemi‐conservaCve,bidirecConal

andsemi‐disconCnuous2. Helicases,DNAgyraseandSSBareneededto

prepareaDNAforreplicaCon.3. RNAprimasecreatesprimerstostartDNA

replicaCon.4. DNAissynthesizedfromapre‐exisCng3’‐OHinthe

5’‐3’direcConbyDNApolymerases(withproofreadingmechanism)

5. DNAligaseconnectsfragments6. DNAisshortenedagerreplicaCon,toprevent

adverseeffects,telomeresarepresent.7. DNAscanbesynthesizedfromRNAtemplatevia

reversetranscriptase.

DNADAMAGEANDREPAIR

41

42

ChangesinthebasesequenceofDNAarecalledmutaGons.TheymayhaveadverseeffectonthenormalbiologicalfuncConsorsilent.MutaConsmaybespontaneousorinduced.

ThegenedefectisaknownmutaConofasinglenucleoCde(TtoA)oftheβ‐globingene,whichresultsinglutamatebeingsubsCtutedbyvalineatposiCon6.

hkp://www.blackpast.com/files/blackpast_images/Sickle_Cell.jpg

43

SpontaneousmutaConsarethosethatoccurduringnormalgeneCcandmetabolicfuncCons.

1. SubsCtuConofbasepaira.  TransiConb.  Transversion

2. DeleConofbasepair/s3. InserConofbasepair/s

44

Nitrogeneousbasesmaybemodifiedbynaturallyoccuringspecies(water)andreacCveoxygenspecies(ROS).

RepairedbyDNAglycosidasescleavethenitrogeneousbases

45

EnvironmentalfactorsalsocausemutaCons.Thesearecalledmutagens.OnetypeofthismutagenareionizingradiaCons.

46

ChemicalAgentscanmodifyDNA:(1)Heterocyclicbaseanalogs,(2)ReacCvechemicals,(3)IntercalaCngagents

5‐BUisanexampleofheterocyclicbaseanalogs.

47

ChemicalAgentscanmodifyDNA:(1)Heterocyclicbaseanalogs,(2)ReacCvechemicals,(3)IntercalaCngagents

 Nitrousacid(HNO2)  Convertsadeninetohypoxanthine,cytosinetouracil,andguaninetoxanthine

  CausesA‐TtoG‐CtransiCons

48

ChemicalAgentscanmodifyDNA:(1)Heterocyclicbaseanalogs,(2)ReacCvechemicals,(3)IntercalaCngagents

49

ChemicalAgentscanmodifyDNA:(1)Heterocyclicbaseanalogs,(2)ReacCvechemicals,(3)IntercalaCngagents

  IntercalaCngagentsareflataromaCccompound.CausesinserConordeleCon

50

DNAsrepairdamagevianuceloCdeexcisionorbaseexcision

51

DNAsrepairdamagevianuceloCdeexcision

52

DNAsrepairdamagevianuceloCdeexcision

53

DNAsrepairdamagevianuceloCdeexcision

RECAP:1.  IntegrityofDNAcanbejeopardizedduring

replicaCon.2. Damagemayalsocomeintheformofnatural

reacConsfromtheenvironment:viachemicalorradiaCon

3. DNArepairsitselfviabaseexcisionornucleoCdeexcisionprocesses

TRANSCRIPTIONOFDNATORNA

55

56

MechanismofRNAsynthesisissimilartoDNAsynthesis.

  RNAPolymerases  Template(DNA) AcCvatedprecursors(NTP) Divalentmetalion(Mg2+orMn2+)

57

MechanismofRNAsynthesisissimilartoDNAsynthesis.

DNAcodingstrand

DNAtemplate(SENSE)strand

5’

3’ 5’

3’

DNAcodingstrand

DNAtemplatestrand

5’

3’ 5’

3’

mRNA5’ 3’RNAPol

5’‐GTCCCAATTGGATGCCCTAGCTCGCTAGAATAGAGATATAGAGATAAACC‐3’

5’‐GUCCCAAUUGGAUGCCCUAGCUCGCUAGAAUAGAGAUAUAGAGAUAAACC‐3’

5’‐GTCCCAATTGGATGCCCTAGCTCGCTAGAATAGAGATATAGAGATAAACC‐3’

3’‐CAGGGTTAACCTACGGGATCGAGCGATCTTATCTCTATATCTCTATTTGG‐5’

3’‐CAGGGTTAACCTACGGGATCGAGCGATCTTATCTCTATATCTCTATTTGG‐5’

58

MechanismofRNAsynthesisissimilartoDNAsynthesis.

59

DifferenttypesofRNApolymeraseencodefordifferenttypesofRNA.MostcommonofwhichisRNAPolymeraseII

60

IntrinsicmarkersintheDNAmoleculedirectstranscripConenzymeswheretostart.Thesearecalledpromotersites.

61

RNApolymeraseakachtoDNAandstartcreaCngRNAPrimarytranscripts.NOTE,noprimerisneeded!

62

Ineukaryotesthealphasubunitisnotasimplecompound,butratherLOTSoftranscripGonfactorswhichhavevariedrolesinpromoCngandcontrollingtranscripCon

63

RNApolymeraseakachtoDNAandstartcreaCngRNA.NOTE,noprimerisneeded!

64

RNApolymeraseakachtoDNAandstartcreaCngRNA.NOTE,noprimerisneeded!

65

AterminatorfactorrecognizesspecificDNAsequencesandsignalsRNAPoltostopencodingRNAfromtheDNAandstartcreaCngaterminalsequence

InE.coliitcreatesahairpintailwithmanyUracils

66

Ineukaryotes,apoly‐Atailissynthesizedbypoly‐Apolymeraseandpoly‐AbindingproteinsagerterminaConofRNAsequence

67

RNAcanalsobesynthesizedfromfellowRNA(viaRNAreplicase).

68

DifferentkindsofRNAsrequiredifferentposttranscripConmodificaCons

RNA

mRNA tRNA

MethylatedGTPcapPolyadenylaConSplicing

BasemodificaConAcetylaCon

69

PrimarytranscriptsfortRNAproducConarecutupinseveralplacesbynucleases.

Nitrogenousbasesaremodified(mostlymethylated)

Aminoacylgroupisaddedonthe3’‐OHend

70

RNAshaveatriphosphateatthe5’end,whichiscappedbyGMPandthenmethylated

CappingensuresthefateofanmRNAasacodecarryingmolecule.(ItallowsthebodytodisCnguishmRNAfromothertypesofRNA)

71

PrimarytranscriptsformRNAcontainingnon‐codingregionscalledintrons.

  Introns Interveningsequences

  Exons Expressedsequences

72

Pre‐mRNAscontainingnon‐codingregionscalledintrons.

73

SplicingdefectscancauseseriousmedicalimplicaCons

  Figure28.28.SplicingDefects.MutaConofasinglebase(GtoA)inanintronoftheb‐globingeneleadstothalassemia.ThismutaCongeneratesanewsplicesite(blue)akintothenormalone(yellow)butfartherupstream.

74

Somepre‐mRNAmoleculescanbesplicedinalternaCvewaystoyielddifferentmRNA

Apre‐mRNAwithmulCpleexonsissomeCmessplicedindifferentways.Here,withtwoalternaCveexons(exons2Aand2B)present,themRNAcanbeproducedwithneither,either,orbothexonsincluded.MorecomplexalternaCvesplicingpakernsalsoarepossible.

75

Somepre‐mRNAmoleculescanbesplicedinalternaCvewaystoyielddifferentmRNA

AlternaCvesplicingprovidesapowerfulmechanismforexpandingtheversaClityofgenomicsequences.

AlternaCvesplicingprovidesanopportunityforcombinatorialcontrol.

RECAP:1. RNAsynthesisissimilartoDNAexcept

a.  RNApolymeraseisdirectedbytranscripConfactors

b.  RNAsdonotneedaprimertostarttranscripCon

c.  Thereisnoproof‐readingmechanismd.  Post‐transcripConmodificaConsarenecessary:

capping,poly‐Atail,splicing