How Cells Read the

Genome: From DNA to

Protein
PROTEIN
translation
Transcription
Prokaryotes Eukaryotes
TranscriptioninProkaryotes
Transcriptionis
catalyzedbyRNA
polymerasewhich
synthesizesRNA
usingDNAas
template
Bacterialcellshavesingle kindof
RNApolymerasethatsynthesizes
allthreemajorclassesofRNA
mRNA,tRNA andrRNA.
ProkaryoticRNApolymerasehastwo
forms:
i.Coreenzyme and
ii.Holoenzyme
Thecoreenzymeisatetramer which
composedof
2
(twoalphasubunits,
onebetasubunit,andonebetaprime
subunit).
CoreRNApolymeraseiscapableof
faithfullycopyingDNAintoRNAbut
doesnotrecognizethepromoter
specifically.
Correctpromoterrecognitionisthe
functionoftheholoenzyme formof
RNApolymerase.
TheRNApolymeraseholoenzyme
contains (sigma)subunit,inadditionto
thesubunitsfoundinthecoreenzyme.
Holoenzyme (
2
+),iscapableof
correctinitiationatthepromoter
regionofagene.
Sigmathusinvolvedinpromoter
recognition.
Transcriptioninvolvesfourstages:
(i)Binding
(ii)Initiation
(iii)Elongation
(iv)Termination
(1)BindingofRNApolymerasetoapromotersequence
RNApolymeraseholoenzyme startsbyrecognizing
thepromoter tosynthesizesRNAfromthecoding
regionofthegene.
A promoter isaregulatoryregionof
DNAlocatedupstream(towardsthe5'
region)ofageneandcontainsspecific
DNAsequencesthatarerecognizedby
RNApolymeraseholoenzyme.
E.coli promoterhastwoconservedregionsnear
positions35and10relativetothetranscription
startsite.
RNAsynthesisbegins35basepairsdownstreamof
thefirstconsensusregionandtenbasepairs
downstreamofthesecond.
Pribnow Box 35element
BindingofRNApolymeraseto
thepromoterinvolvesashort
unwinding oftheDNAdouble
helix.Thisisaccomplishedina
twostepfashion.
First,RNApolymeraseformthe
closedcomplex,whichis
relativelyweak.Then,the
doublestrandedDNAgoes
throughaconformational
changetoformthemuch
stronger opencomplex.
(2)InitiationofRNAsynthesis
UnwoundoftheDNAdoublehelix,leadstoinitiation
ofRNAsynthesis.
OneofthetwoexposedsinglestrandedDNAserves
asthetemplateforthesynthesisofRNAusing
ribonucleoside triphosphate molecules(NTPs)as
substrates.
AssoonasthefirstNTPformshydrogenbondwith
thecomplementarybaseofthetemplateDNA
strand,RNApolymerasecatalyzestheformationof
phosphodiester bondbetweenfirstandsecondNTP
(3)Elongation
ChainelongationcontinuesasRNA
polymerasemovesalongtheDNA
molecules,unwindingthehelixbitbybit
andaddingonecomplementarynucleotide
tothegrowingRNAchain.RNAstrand
elongatesinthe53 direction.Theaverage
speedoftranscriptionisabout40
nucleotidespersecond.
(4)TerminationofRNAsynthesis
Twoterminationmechanismsarewellknown:
(i)Rhodependenttermination
(ii)Rhoindependenttranscriptiontermination
orIntrinsictermination
(i)Rhodependenttermination
Itusesa terminationfactor called factor(rho
factor)whichisaproteintostopRNAsynthesisat
specificsites.
Thisproteinbindsatarho
utilisation site onthenascent
RNAstrandandrunsalong
themRNAtowardstheRNA
Polymerase.
Astemloopstructure
upstreamoftheterminator
regionpausestheRNA
Polymerase,whenfactor
reachestheRNApolymerase,
itcausesRNApolymeraseto
dissociatefromtheDNA,
terminatingtranscription.
(ii)Rhoindependenttranscription
Rhoindependenttranscriptionterminationinvolves
terminatorsequenceswithintheRNAthatsignalthe
RNApolymerasetostop.Theterminatorsequenceis
usuallya palindromic sequencethatformsastem
loop hairpin structurethatleadstothedissociation
oftheRNAPolymerasefromtheDNAtemplate.
Thesesequencescaneither(a)AstrongGCrichstem
andloop,(b)asequenceof46UresiduesintheRNA,
whicharetranscribedfromacorrespondingstretchof
asinthetemplate.
ProkaryoticTranscription
TranscriptioninEukaryotes
Eukaryotestranscriptiondiffers
inseveralrespectsfromthatof
prokaryotes.
Inprokaryotes(bacteria),transcriptionoccursinthe
cytoplasmwhereasineukaryotes,transcriptionoccursin
thecell'snucleus,mRNAthenmovestothecytoplasm
for translation.
Transcriptionineukaryotesismore
complexbecausethesizeofgenome
is1000timesgreaterthan
prokaryotesanditinvolvesseveral
proteinfactors.
DNAinprokaryotesismuchmore
accessibletoRNApolymerasethanDNAin
eukaryotes.
EukaryoticDNAiswrappedaround
proteinscalledhistones toformstructures
callednucleosomes.EukaryoticDNAis
packedtoformchromatin.
mRNAproducedasaresultof
transcriptionisnotmodifiedin
prokaryoticcells.Eukaryoticcells
modifymRNAbyRNAsplicing,5'
endcapping,andadditionofa
polyA tail.
EukaryotictranscriptionusesthreedistinctRNA
polymerases, RNApolymeraseI, RNApolymeraseII
and RNApolymeraseIII, eachspecializedfordifferent
RNAs.
Themostusefulcriterionfordistinguishingthe
polymeraseistheirsensitivitytoinhibitionby
amanitin,theactivecompoundinthepoisonous
mushroom Aminita phalloides, ordestroying
angel.
RNApolymeraseI islocalizedinthenucleolus andnot
inhibitedbyamanitin.RNApol Itranscribesthelarge
ribosomalRNAs (5.8S,18S,28SrRNAs).
RNApolymeraseII islocalizedinthenucleoplasm
andinhibitedatverylowconcentrationsof
amanitin (0.1g/ml).RNApol IItranscribesthe
messengerRNAs (mRNA)andmostsmallnuclear
RNAs (SnRNAs).
RNApolymeraseIII isalsolocalizedinthe
nucleoplasm andinhibitedbyveryhigh
concentrationsofamanitin (10g/ml). RNApol III
transcribestransferRNAs (tRNA),5SribosomalRNA
and7Ssmallcytoplasmic RNA(scRNA).
Eukaryotictranscriptionisdependentonseveralsequenceand
structuralfeatures.
(i)Activelytranscribinggeneshavealooser, moreaccessible
chromatinstructure.Thenucleosomes arenotascondensedasinother
formsofchromatin,especiallyheterochromatin,andtheyoftendonot
containhistone H1.TheDNAinthepromoter regionatthe5 endof
thegenemaynotbeboundintonucleosomes atall.Inthisway,the
promotersequencesareavailableforbindingtoproteintranscription
factors.
(ii)Inadditiontopromotersequences,othernucleotide
sequencestermedenhancers canaffecttranscription
efficiency.Unlikepromoters,whichonlyaffectsequences
immediatelyadjacenttothem,enhancersfunctionevenwhen
theyarelocatedfaraway(asmuchas1,000basepairsaway)
fromthepromoter.
(iii)Bothenhancerbindingandpromoterbinding
transcriptionfactorsrecognizetheirappropriateDNA
sequencesandthenbindtootherproteinsforexample,RNA
polymerase,tohelpinitiatetranscription.Becauseenhancers
arelocatedsofarfromthepromoterswhereRNApolymerase
binds,enhancerinteractionsinvolvebendingtheDNAto
makealoopsotheproteinscaninteract.
RibosomalRNAsynthesis
RibosomalRNAsynthesis
MostoftheRNAmadeinthecellisribosomalRNA.
RibosomalRNAismadeinthenucleolus,whichcontains
manycopiesoftherRNA genes.The28Sand5.8Slarge
subunitRNAs andthesmallsubunit18SRNAare
synthesizedbyRNApolymeraseIfromasinglepromoter.
Thesegenesexistintandemcopies(severalhundredrepeats)
oneaftertheotherintheorder:28S5.8S18S,separated
bynontranscribed spacersandorganizedinacluster.The45S
preRNAtranscriptswhichformsarelaterreleasedby
cleavagesandprocessing.
Thepromoterhasbeenbestcharacterizedinhumancells,
inwhichitconsistsofabipartitesequenceintheregion
precedingthestartpoint.Thecorepromotersurroundsthe
startpoint,extendingfrom45to+20,andissufficientfor
transcriptiontoinitiate.However,itsefficiencyisverymuch
increasedbytheupstreamcontrolelement(UCE),which
extendsfrom180to107.Bothregionshaveanunusual
compositionforapromoter,beingrichinGCbasepairs;
andtheyareabout85%identical.
Initiation
RNApolymeraseIrequires
two ancillaryfactors.
UBF1 (upstream binding factor 1)
is a single polypeptide that binds
to a GCrich element in the core
promoter and UCE. Factor SL1
(selectivity factor 1) does not by
itself have specificity for the
promoter, but once UBF1 has
bound, SL1 can bind cooperatively
toextendtheregionofDNAthatis
covered. Once both factors are
bound,RNApolymeraseIcanbind
to the core promoter to initiate
transcription.
Elongation
AsPol Iescapesandclearsthepromoter,
UBFandSL1remainpromoterbound,ready
torecruitanotherPol I.Indeed,eachactive
rDNA genecanbetranscribedmultipletimes
simultaneously.
Termination
TranscriptionterminationofRNApolymeraseI
(Pol I)isamultistep processinvolvingPol I
pausing,releaseand3 endprocessingofthe
primarytranscript.Althoughthesequencesof
theterminatorelements/siteandtheDNA
bindingproteinsvariesfromspeciestospecies
butthemechanismofterminationinall
eukaryotesissimilar.
Inhighereukaryotes,terminationfactorTTFI
bindsandbendstheterminationelements/siteat
the3'endofthetranscribedregion.
Inmouse,an18bp Sal box consensusmotif,
AGGTCGACCAGA/TT/ATCCG,hasbeenshownto
bindtheterminationfactorTTFI.ThisforcePol I
topause.TTFI,withthehelpoftranscript
releasefactorPTRFandaTrichregion,will
inducePol Iintoterminatingtranscriptionand
dissociatingfromtheDNAandthenew
transcript.
MessengerRNAtranscription
MessengerRNAtranscription
RNApolymeraseIItranscribesmessengerRNAandafew
othersmallcellularRNAs.Twobasalpromoterelements
thatarefoundinessentiallyalleukaryoticmRNAgenes
aretheTATAboxandtheCAATbox.Theseelementsare
socalledbecauseoftheDNAsequencesthatconstitute
thepromoterelement.TheTATAboxisfound
approximately2025basesupstreamofthestartsitefor
transcriptionandtheCAATboxisaround100bases
upstream.
LikeRNApolymerasesI,polymeraseIIcannot
actalone.Instead,manytranscriptionfactor
assembleonpromoterDNAwithpolymeraseII,
creatingalargemultiproteinDNAcomplex
thatsupportsaccurateinitiation.These
transcriptionfactors(TFIIA,TFIIB,TFIID,TFIIE,
TFIIF,andTFIIH)controltheactivityofRNApol
IIandthus,thenomenclatureoftheseproteins
isTFII,fortranscriptionfactorofRNApol II.
TFIIDisthefactorthatbindstotheTATAbox
anditsbindingisfacilitatedbyTFIIA.Once
TFIIDandTFIIAareboundTFIIBbindsandthis
recruitsRNApol IItothepromoter.NextTFIIE,
TFIIFandTFIIHbindtoinitiatetranscription.
Elongation
The speed of the Pol II elongation complex is around ~14
kb per min depends on an interactions between polymerase
and nucleic acids (DNA and RNA) and base-pairing
interactions between single-stranded RNA and DNA.
Termination
Twotypesofmechanismhavebeen
reportedfortranscriptiontermination
ofRNApolymeraseII.
1.Poly(A)dependenttermination.
2.Sen1dependenttermination.
Inpoly(A)dependenttermination inyeast,the53
exoribonuclease RNAtraffickingprotein1(Rat1)interact
withpoly(A)siteRNAelements(ArichandUrich
sequences).Rat1thendegradestheRNAthatresultin
disassociationofthePol II.
InSen1dependenttermination inyeast,Sen1is
bindstoPol IIviaNrd1proteinsandinteractwith
specificRNAelements(GUAArepeats).Sen1unwind
theDNARNAhybridviaitshelicase activityandthus
terminatethetranscriptionprocess.
Transferand5SribosomalRNAtranscription
Transferand5SribosomalRNAtranscription
RNApolymeraseIIItranscribes5SrRNA andtRNA genes.
Thepromotersfor5SandtRNA genesareinternal;theylie
downstreamofthestartpoint.ThepromotersforsnRNA
(smallnuclearRNA)geneslieupstreamofthestartpoint.
Thepromoterfor5SRNAtranscriptionliesbetween
positions+55and+80withinthegene.Three typesof
transcriptionfactorsi.e.,TFIIIA ,TFIIIB and TFIIICare
involvedthatrecognizestheinternalpromoters.
Attype2promoters,TFIIICrecognizesboxB,butbindstoamore
extensiveregionincludingbothboxesAandB.Attype1promoters,
TFIIIAbindstoasequencethatincludesboxC,andthisisrequiredto
enableTFIIICtobind.Inbothcases,thebindingofTFIIICinturn
enablesTFIIIBtobindtoasequencesurroundingthestartpoint.
TFIIIBfunctionsasa"positioningfactor,"responsibleforlocalizing
RNApolymeraseIIIcorrectly.
Theupstreamregionhasamoreimportantrolein
thethirdclassofpolymeraseIllpromoters,where
therearethreeupstreamelementsTATAelement,
PSE(ProximalSequence Element)andOctelements.
Thefactorsthatbindattheseelementsinteract
cooperatively.Theboundfactortheninteractswith
RNApolymeraseIII,whichisthencapableof
initiation.
RNApolymeraseIIIterminatestranscriptionwithU's
immediatelyafteraGCrichregion.