Professional Documents
Culture Documents
RNAPolymerasesandGeneral
TranscriptionFactors
Nucleicacidpolymerases:classification
Nucleicacidpolymerases
Sugarspecificity
AddsrNTPs
AddsdNTPs
RNApolymerases
DNApolymerases
Templatespecificity
DNAdependent
RNAdependent
DNAdependent
RNAdependent
RNApolymerases
RNApolymerases
DNApolymerases
DNApolymerases
RNApolymeraseII
HCVreplicase
E.coliDNA
polymeraseIII
HIVreverse
transcriptase
SimpleandcomplexDNAdependentRNA
polymerases
ViralRNApolymerases(1subunit)
ProkaryoticRNApolymerases(4different
subunits)
EukaryoticandarchealRNApolymerases
(atleast12subunits)
Viruses
MultipleFormsofEukaryoticRNAPolymeraseEarly
studies
There are at least two RNA polymerases operating in
eukaryoticnuclei
OnetranscribesmajorribosomalRNA(rRNA)genes
Oneormoretotranscriberestofnucleargenes
Ribosomalgenesaredifferentfromothernucleargenes
Differentbasecompositionfromothernucleargenes
Unusuallyrepetitive
Foundinthenucleolus
SeparationoftheThreeNuclear
Polymerases
EukaryoticnucleicontainthreeRNApolymerases
Separatedbyionexchangechromatography
RNApolymeraseIfoundinnucleolus
transcribesrRNAgenes
RNApolymerasesIIandIIIarefoundinthe
nucleoplasm
transcribesotherkindsofRNA
RolesofthreeRNAPolymerases
Polymerase I makes large
rRNAprecursor
PolymeraseIImakes
Heterogeneous nuclear RNA
(hnRNA)
mRNA
SmallnuclearRNA
3RNApolymerasesineukaryotes
Name
Makes amanitin
RNA
prerRNA insensitive
PolymeraseI
RNA
premRNA verysensitive
PolymeraseII somesnRNAs
RNA
pretRNA lesssensitive
PolymeraseIII
othersmallRNAs
somesnRNAs
Subunitstructureofeukaryotic
RNApolymerases
All3havemultiplesubunits(8to14).
MWforeachpolymeraseisabout500,000
daltons
Somesubunitsarecommontoall3RNA
polymerases
All3RNApolymeraseshavesubunitsthatare
homologoustothebacterial,and
subunits.
DistinctformsofRNApolymeraseusedfor
initiationandelongation:RNAPolII
EukaryoticRNApolym
eraseI
PolIa kinase+ATP
PolIo
phosphatase P
CTDoflargesubunitofPolI
P
P
P
P
CTDoflargesubunitofPolI
P
CTDhasrepeatof(YSPTSPT) .
2650
CTD=Cterminaldomain
Model:PhosphorylationofPolIIatomakePolIIois
neededtoreleasethepolymerasefromtheinitiation
complexandallowittostartelongation.
PolymeraseII
Young10subunitsareplacedin3groups:
Core(3ofthesubunits)relatedinstructure
andfunctiontobacterialcoresubunits
Common(5ofthesubunits)foundinall3
nuclearRNApolymerasesinyeast
Nonessential subunits (2 of the subunits)
conditionally dispensable for enzymatic
activity
CoreSubunits
Three polypeptides Rpb1, Rpb2, Rpb3 absolutely
requiredforenzymeactivity
Thesearehomologousto,,andsubunits
BothRpb1andsubunitbindsDNA
Rpb2 and subunit are at or near the nucleotide
joiningactivesite
Rpb3doesnotresemblesubunit
Thereisone20aminoacidsubunitofgreatsimilarity
2subunitsareaboutsamesizesamestoichiometry
CommonSubunits
Therearefivecommonsubunits
Rpb5
Rpb6
Rpb8
Rpb10
Rpb12
Littleknownaboutfunction
Theyareallfoundinall3polymerases
Suggestsplayrolesfundamentalintranscription
SubunitsNonessentialforElongation
Rpb4andRpb7
Dissociatefairlyeasilyfrompolymerase
Might shuttle from one polymerase II to
another
Rpb4mayhelpanchorRpb7totheenzyme
Mutants without Rpb4 and Rpb7 transcribes
wellbutcannotinitiateatarealpromoter
Rpb7isanessentialsubunit
TheThreeDimensionalStructureof
RNAPolymeraseII
StructureofyeastpolymeraseII(polII4/7)revealsa
deepcleftthatacceptsalinearDNAtemplatefromone
endtoanother
Catalyticcenterliesatthebottomofthecleftandcontains
aMg2+ion
UpperjawRpb1+Rpb9andlowerjawRpb5
Geometryallowsenoughspacefor:
TFIIDtobindattheTATAboxofthepromoter
TFIIBtolinkthepolymerasetoTFIID
Placespolymerasecorrectlytoinitiatetranscription
PositionofNucleicAcidsinthe
TranscriptionBubble
DNAtemplatestrandis
showninblue
DNAnontemplatestrand
showningreen
RNAisshowninred
PositionofCriticalElementsinthe
TranscriptionBubble
Three loops of the transcription
bubbleare:
Rudder: initiating RNA
DNAdissociation
Lid: maintains RNADNA
dissociation
Zipper:
dissociation
DNA
maintaining
of template
Transcriptionmechanism
Pore1alsoappearstobe
theconduitfor:
Nucleotides to enter the
enzyme
RNA to exit the enzyme
during
backtracking
(pausing and error
correction)
Theeukaryoticpromoters
ClassIIpromoters
Class II Promoters
recognized by RNA
polymerase II are
similar to prokaryotic
promoters
Considered to have two
parts:
Core promoter having 4
elements
Upstream
promoter
element
CorePromoterElementsTATABox
Foundonthenontemplatestrand
Verysimilartotheprokaryotic10box
TherearefrequentlyTATAlesspromoters
Housekeeping genes that are constitutively
active in nearly all cells as they control
commonbiochemicalpathways
Developmentallyregulatedgenes
TheTATAbox
Otherpromotersequences
Initiator(Inr):YYA+1NT/AYYY(Tyrosinerich).
SomepromoterscontainbothinitiatorandTATAbox,
whereasothersonlyoneofthem
CpGislands:CGrichstretchof2050nucleotideswithin
~100basepairsupstreamthestartsite.
BRE(TFIIBrecognitionelement)sequence,whichhasthe
consensusG/CG/CG/ACGCC,islocatedimmediately
upstreamoftheTATAelementofsomepromotersand
increasestheaffinityofTFIIBforthepromoter
Downstreampromoterelement(DPE)ispresentinsome
geneswithinitiatorpromoter.Itislocatedabout
30nucleotidesdownstreamofthetranscriptionstartsite
andincludesacommonGA/TCGsequence
Upstreampromoter
Upstreampromoterelementsareusuallyfound
upstreamofclassIIcorepromoters
Differfromcorepromotersinbindingtorelatively
genespecifictranscriptionfactors
GCboxesbindtranscriptionfactorSp1
CCAATboxesbindCTF(CCAATbinding
transcriptionfactor)
ClassIpromoters
ClassIpromotersarenotwellconservedin
sequenceacrossspecies
Generalarchitectureofthepromoteriswell
conservedtwoelements:
Coreelementsurroundingtranscriptionstartsite
Upstreampromoterelement(UPE)100bpfarther
upstream
Spacingbetweentheseelementsisimportant
ThreetypesofclassIIIpromoters
TypeI(5SrRNA)has3
regions:
BoxA
Shortintermediateelement
BoxC
TypeII(tRNA)has2regions:
BoxA
BoxB
TypeIII(nonclassical)
resemblethoseoftypeII
Summaryofpromoterelements
CpGisland
approx.100to1
EnhancersandSilencers
These are position and orientationindependent
DNA elements that stimulate or depress,
respectivelytranscriptionofassociatedgenes
Areoftentissuespecificinthattheyrelyontissue
specificDNAbindingproteinsfortheiractivities
Some DNA elements can act either as enhancer or
silencerdependingonwhatisboundtoit
Generaltranscriptionfactors
General transcription factors are required for
transcriptionineukaryotesfromallgenes
GTFsassistRNAPolIIintranscriptioninitiation
GTFs are designated TFIIA, TFIIB,... and most
ofthemaremultimericproteins
Equivalent GTFs are highly conserved among
theeukaryotes
In prokaryotes, only one general transcription
factor,knownasfactorisrequired
TFIID
TFIIDiscomposedof14subunits,oneofthembeing
TATAboxbindingprotein,TBP
Functions:promoterrecognition,TFIIBrecruitment
TheTATAboxbindingprotein(TBP)
Bindstominorgroove
SummaryofinteractionsbetweenTBPand
TATAbox
Phe190
TATAboxismakingapseudo
twofoldsequencespecific
interactionwithtwothreonines
andtwoasparaginesofTBP
Stackingofphenylalanines
againstDNAbasesisalso
shown(DNAbendingdueto
stackingisnotshown)
Phe99
TFIIB
Functions:
startsiteselectionforPolII
TFIIFPolIIcomplexrecruitment
SomeTFIIBmutantsresultinashiftoftranscriptionstartsite
Under certain conditions (BRE element promoter) Pol II together with
TFIID and TFIIB can form the minimal initiation complex. At most
promoters,however,TFIIE,FandHarenecessary
Cterminaldomain(CTD)ofTFIIB
CTD of TFIIB interacts with
both TBP and DNA around
the promoter especially
BREelement
RoughpositioningofPolIIis
due to interaction of TFIIB
CTDwithTBP
Fine positioning is due to
interactionwithDNA
Nterminal domain of TFIIB
interactswithPolII
NTD
PolII
TFIIF
Functions:
RecruitmentofPolIItotheexistingDNATFIIDBcomplex,
PositioningPolIIoverthestartsite
BindingtothenontemplateDNAstrand.
TFIIFalsoreducesnonspecificbindingofRNApolIItoDNA.
The3DstructureofTFIIF
TFIIF
is
a
heterotetramer, made
from two subunits of
RAP30
and
two
subunits of RAP74
proteins
RAP30RAP74 dimer
within the complex
structure has an unusual
triplebarrelfold
TFIIE
TFIIE is a heterotetrameric
protein(2)
Functions:
TFIIE appears to create the
docking site for next
transcriptionfactor,TFIIH.
TFIIE also modulates TFIIH
enzymaticactivities
In addition TFIIE enhances
promotermelting.
TFIIH
Earlyeventsin
elongation
AsPolIItranscribesawayfromthestartsitesubunitof
TFIIHphosphorylatesthePolIICTD,whichresultsin
promoterescape.
Generalfactorsgetreleased
TFIIA
Fortranscriptioninvivo,anotherfactorTFIIAis
required
ThefunctionofTFIIAissomewhatunclear,butit
mighthelptheotherfactorstobind.
TFIIAhasalsoshowntohavesomeantirepressor
functions
TFIIAisnotrequiredfortranscriptioninvitro.
TAFs
ApartfromTBP,TFIIDhas13TAF(TBP
associatedfactors)subunits
Some of them seem to be necessary for
transcription initiation from promoters,
lackingtheTATAbox
OtherTAFshavebeenshowntobetissue
specificcoactivators
TAFsubunitsalsointeractwithotherGTFs
thereforestabilyzingthecomplex.
The3DstructureofPolIIholoenzyme
TBPlikeproteins
(TLFs)
TBPlikeproteinsareTBP
proteinanalogues(nottobe
confusedwithTAFs)
SomeTLFsreconize
TATAbox
OtherTLFsrecognizesome
differntpromotersequence
TLFsarethoughttoplaya
roleintranscription
regulation
TLFsastranscriptionregulators
WhatisPolIIbacksliding,pausingandarrest?
Backsliding is an event when RNA polymerase moves
backwards and newly made RNA gets inserted in the
funnel.ItcanbecausedbyincorporationofwrongNTPor
when3OHloosescontactwithactivesiteMg2+
If the backsliding RNA piece is 24 nt long, this a
reversible process and RNA polymerase can recover by
itself.Thisiscalledpausing.
If the backsliding RNA gets longer (710 nt), it gets
trapped in funnel pore and RNA polymerase can not
recoverbyitself.Thisiscausedarrest.
Arrest can be overcome by specific elongation factors,
whichhelpRNAPolIItocleavethearrestedRNA
TranscriptionelongationfactorIIS
Elongatedshapewhy?
TFIISgetsinsertedinactivesiteofPolII
Domain III of TFIIS gets inserted in the
activesiteofPolII
DomainIIdocksonthesurface
InteractionofTFIISacidic
hairpinwitharrestedRNA
THeacidicresidues
D290andE291
assistincleavingthe
phosphodiesterbond
Thebacktracked
RNAfragmentgets
releasedandRNA
PolIIisrescued
fromarrest
Elongatordecondensesthe
chromatin
Otherelongationfactors
PTEFb (Positive Transcription Elongation Factor)
phosphorylates additional residues in CTD of PolII
(somearealreadyphosphorylatedbyTFIIH).
ELL family of proteins also stimulates transcription
elongation through a poorly understood mechanism.
There is some evidence, that ELLs target RNA
polymerase for degradation upon reaching damaged
regions in DNA. Degradation of PolII is followed by
recruitmentofDNArepairingenzymes.
Negative factors NELF and DSIF supress elongation
bydirectinteractionwithPolII
Othereukaryotictranscription
systems
RNApolI(prerRNA)
RNApolIII(tRNA,5SrRNA)
MitochondrialRNApolymerase
ChloroplastRNApolymerase
RNAPolI
RNApolIII
Unlikeotherpolymerases,promoter
sequenceslieentirelywithincoding
sequence.TheconservedAandBsites
alsocodefortwoinvariantsequences,
observedinalltRNAs
ThreeinitiationfactorsarereqiredforPolIII
initiation.TFIIIAisrequiredonlyfor5SrRNA
transcription.TFIIIBhasthreesubunits,oneof
themsimilartoTFIIBandanotheronebeing
TBP(sameasforPolIandII)
Eukaryotic
Transcription
Overview
Introduction
Transcription in Prokaryotes and
Eukaryotes
Pre-Initiation
Initiation
Elongation
Termination
Post- Transcription
InProkaryotes&Eukaryotes
Prokaryotes
Eukaryotes
Occursincytoplasm
Coupledtranscription&
translation
Nodefinitephaseof
occurrence
AsingleRNAPsynthesizes
mRNA,tRNA&rRNA
Noinitiationfactors
required
Occursinnucleus
Nocouplingoftranscription&
translation
OccursintheG1&G2phases
RNAPI,II&IIIsynthesize
rRNA,mRNA&tRNA
TFIIA,TFIIB,TFIID,TFIIE,
TFIIF,TFIIHrecognizeTATA
box
Monocistronic
Polycistronic
Polycistronic&Monicistronic
Monocistronic
AnmRNAmoleculeissaidtobe
monocistronic if it contains
genetic information to translate
onlyasingleprotein.
Intheendonlyonepolypeptide
chain coding for one protein is
obtained from a single gene
with an operator & promoter
region.
Polycistronic
Polycistronic mRNA contains
information for several genes
which are translated into
severalproteins.
mRNA contains several ORFs
(open reading frames), each of
which is translated in proteins.
Thecodingisgroupedandallof
the genes are translated
together with a common
promoter & operator region
(likeinoperons)
Pre-Initiation
First step of transcription.
The Pre-Initiation Complex (PIC)
includes RNA Polymerase II and
six transcription factors- TFIIA,
TFIIB, TFIID, TFIIE, TFIIF, TFIIH
Other co-activators and
chromatin remodeling
complexes also comprise of PIC
Pre-Initiation
(contd.)
Can be summarized
in 3 steps:
1.TATA Binding Protein (TBP) is a
subunit of TFIID and binds to the
promoter, creating a sharp bend.
2.TBP-TFIIA interact; TBP-TFIIB interact;
TFIIB-TFIIF interact & TFIIF recruits
RNA Pol II; TFIIE joins the group and
recruits TFIIH
3.Subunits within TFIIH that
haveATPaseandhelicaseactivity
create negativesuperhelicaltension
in the DNA.
Initiation
1. Negative superhelical tension causes
approximately
one
turn
of
DNA
tounwindand form thetranscription
bubble. Promoter melting requires
hydrolysis by ATP and is mediated by
TFIIH.
2. TFIIH pulls the double stranded DNA
into the cleft of RNA Polymerase and
helps in transition from closed to open
state. The two strands get separated.
Initiation
Abortive Initiation
Before entering elongation phase, The
polymerase may terminate prematurely.
This produces a truncated polypeptide
chain.
Many cycles of abortive initiation may
occur before actually producing a
growing polynucleotide chain.
This helps in providing a scrunching
kind of motion.
Elongation
Transcription Fidelity
RNA
polymerases
select
correctnucleoside
triphosphate(NTP)
substrate
to
prevent transcription errors. Only
the NTP which correctly base pairs
with the coding base in the DNA is
admitted to the active center.
RNA polymerase performs two
known proofreading functions to
detect and remove misincorporated
nucleotides:
pyrophosphorylytic
editing and hydrolytic editing
Pausing and
Backtracking
RNA polymerase
does not transcribe
through a gene at a constant pace.
Rather it pauses periodically at
certain sequences, sometimes for
long periods of time before resuming
transcription.
Promoter-proximal pausing during
early elongation is a commonly used
mechanism for regulating genes
poised to be expressed rapidly or in a
coordinated fashion. The blockage is
released
once
the
polymerase
receives an activation signal
Termination
Two Types:
1.Factor Dependent
2.Factor Independent
Factor dependent requires
Termination Factors along with
RNA Pol I.
Factor Independent termination
can be done by RNA Pol III. A
stretch of Thymines along a hair
pin loop causes disintegration of
complexes.
Post
Transcriptional
RNASplicing:
Modifications:
1.
Intronsareremoved
Exonsarejoined
Small nuclear riboproteins (snRNP) like
spliceosomeshelpcatalysethereaction.
Selfsplicingintronsalsoexist.
Mainlyfoundineukaryotes
Post
Transcriptional
Modifications
5 end Capping
2.
A guanine nucleotide linked
to the 5 end triphosphate
3. Polyadenylation
Poly Adenine units added to
3 end of the Ribonucleotide
chain.
Inhibition of
Transcription
Most
antibiotics
are
transcription
inhibitors and are useful against
bacterial and fungal pathogens.
They inhibit action by binding to RNA
Polymerases,
DNA
helicases,
DNA
topoisomerases or by producing free
radicals affecting transcription
For Eg, Rifampicin binds to the Subunit
of RNA Polymerase
CoupledTranscription&Translation
inEukaryotes
Prokaryotic transcription is the process in
whichmessengerRNAtranscripts
of
genetic material in prokaryotes are
produced, to be translated for the
production
ofproteins.
Prokaryotic
transcription occurs in the cytoplasm
alongsidetranslation.
Prokaryotic
transcription and translation can occur
simultaneously. This is impossible
ineukaryotes, where transcription occurs
in a membranebound nucleus while
translation occurs outside the nucleus in
the cytoplasm. In prokaryotes genetic
material is not enclosed in a membrane
enclosed nucleus and has access to
ribosomesinthecytoplasm.