Transcription

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

Polymerase III makes

precursors to tRNAs, 5S
rRNAandothersmallRNA

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)

Bridge helix lies next to

theactivecenter
Flexing this helix may
function in translocation
duringtranscription

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

ATATA boxis a DNA sequence that indicates where a genetic

sequencecanbereadanddecoded.Itisatypeofpromotersequence,
whichspecifiestoothermoleculeswheretranscriptionbegins.

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 is a multimeric protein,

composed of 9 subunits, some of
them with distinct enzymatic
activities
Functions:
TFIIHhasahelicaseactivity,which
unwinds the DNA duplex at a start
site, allowing Pol II to bind to the
templatestrand.
TFIIH also has a kinase ativity, it
phosphorylates PolII in the begining
ofelongation
Other TFIIH subunits have been
shown to recruit DNArepairing
enzymes if polymerase reaches
damaged region in DNA and gets

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

Transcription elongation factors

In vivo rates of transcription = 1200-2000 nucleotides/min.
Baseline in vitro rates = 100-300 nucleotides/min.
In vitro frequent transcription pausing and arrests occur even with chromatinfree DNA
Large number of proteins and protein complexes exist that regulate elongation
of transcription (elongation factors)
Elongation factors display following activities:
Enable elongation through chromatin
Suppress pausing
Overcome transcription arrest

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

Sequential histone acetylation by

transcription-coupled histone
acetyltransferase (part of elongator
complex)

The ebb and flow of

histones
The histone chaperone
activity of Spt6 helps to
redeposit histones on the
DNA thus resetting
chromatin structure after
passage of the large
RNAPII complex

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

The polynucleotide chain is elongated

with the help of Elongation Factors.
RNA Pol conveniently adds nucleotides
to the 3 end. The template strand for
this is known as the sense strand and
the other anti-sense strand.
There are different classes of
elongation factors. Some factors can
increase the overall rate of
transcribing, some can help the
polymerase through transient pausing
sites, and some can assist the
polymerase to transcribe through
chromatin

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.

'Sure. That's the sugar in the DNA,' Smith

said.
'Would it taste sweet?'
'No. DNA is an acid, and it's got salts in it.
Actually, I've never tasted it.'
Later, I got some dried calf DNA. I placed
a bit of the fluff on my tongue. It melted
into a gluey ooze that stuck to the roof of
my mouth in a blob. The blob felt slippery
on my tongue, and the taste of pure DNA
appeared. It had a soft taste, unsweet,
rather bland, with a touch of acid and a
hint of salt. Perhaps like the earth's
primordial sea. It faded away.
Page 67, inRichard Preston'sbiographical
essay onCraig Venter,"The Genome