Elongation and
pre-mRNA processing
RNAPII
MBV4230
Introduction

Themes

main rate-limiting
stages of
transcription
elongation
Stage 1 - first steps - RNA polymerase II is released
 Stage 2 - promoter proximal events - pausing, checkpoint control
 Stage 3 - productive elongation

Chromatin and elongation
 Pre-mRNA processing

Capping, Splicing and Termination/3´-end formation
Odd S. Gabrielsen
MBV4230
Regulation of trx elongation

Regulation of elongation


most transcription units are probably regulated during elongation
because the elongation machinery must coordinate with so many other
nuclear processes while navigating a nucleoprotein template.
Early evidence for general elongation factors

Elongation rate of RNAPII in vitro << in vivo



In vitro: 100-300 nt per min, frequent pauses, some times full arrest
In vivo: 1200-2000 nt per min, probably because elongation-factors
suppress pausing
The DRB-inhibitor: nucleotide-analogue causing strong
inhibition of hnRNA synthesis, acts by enhanced arrest of
RNAPII, but has no effect on purified RNAPII, targets probably
an elongation factor

DRB = 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole
Odd S. Gabrielsen
Elongation stage 1
Promoter escape
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Promoter escape - early steps to
break up contacts to the promoter

ITC


ITC undergoes abortive initiation


ITC cycles through several rounds of abortive initiation, releasing large
amounts of 2–3-nucleotide-long RNA transcripts
Escape commitment


At the onset ITC: the initially transcribing complex (ITC).
After synthesis of the first 4 nucleotides, the B-finger of TFIIB and a
switch domain of Pol II stabilize the short RNA.
Clash with B-finger of TFIIB

After 5 nucleotides are added, the nascent RNA collides with the Bfinger of TFIIB, inducing stress within the ITC.
 probably contributes to the rate-limiting step of promoter escape
 escape: Transition ITC - EEC (early elongation complex)
 Stress from the growing transcription bubble and the production of a 7nucleotide-long RNA trigger collapse of the transcription bubble,
providing the energy to remodel the transcription complex and eject the
5
B-finger from the RNA-exit tunnel and TFIIB is released.
Odd S. Gabrielsen
MBV4230
Stage 1 - the early steps:
from ITC to EEC
6
Odd S. Gabrielsen
Elongation stage 2
Promoter-proximal pausing
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Pausing

pause
RNAPII
Promoter proximal pausing

a phenomenon whereby RNAPII pauses in the 5′ region of the
transcription unit. Requires a signal to proceed.
 Regulatory function: pausing constitutes an important regulation step in
vivo. A stalled RNAPII can escape rapidly from the pause into the
productive elongation phase providing highly dynamic and rapid
response.



Examples: heat-shock-inducible genes and the proto-oncogenes MYC+FOS
Checkpoint control: pausing functions as a checkpoint before
committing to productive elongation.
The Dm hsp70 example

Paused Pol II fully occupies the promoter-proximal region of Hsp70
under conditions in which the gene is not induced. RNAPII paused after
synthesis of about 25 nt of RNA. Pausing mapped to several sites from
8
+20 to +40.
Odd S. Gabrielsen
MBV4230
Several elongation-factors isolated that
control pausing or stimulate elongation
-
P-TEFb
TFIIS
+
NELF
DSIF
+
TFIIF
Elongin
FACT
RNAPII
+
ELL
Odd S. Gabrielsen
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Two negative elongation factors
DSIF and NELF induce the pause

Mechanism of action




NELF = Negative ELongation Factor, multiprot complex (4 polypeptides 46-66 kDa)
DSIF = DRB Sensitivity Inducing Factor, heterodimer (14+160 kDa) = Spt4+Spt5 yeast
In vivo, DSIF and NELF are present at uninduced paused D.m. heat-shock genes.
Stop and wait for capping: DSIF interacts with hypophosphorylated CTD. NELF
recognizes the RNAP II–DSIF complex (may also bind RNA) and halts elongation.
This pause allows the recruitment of the capping enzyme by the CTD and DSIF (Spt5
subunit), which adds a 5´-cap to the nascent transcript.
Odd S. Gabrielsen
Elongation stage 3
Productive elongation
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MBV4230
Release of RNAPII from the pause:
P-TEFb phosphorylates CTD

The P-TEFb (positive transcription elongation factor) complex,
which contains the cyclin-dependent kinase CDK9 and
cyclin T, relieves the negative effects of DSIF and NELF

P-TEFb couples RNA processing to transcription by
phosphorylating Ser2 of the RNAP II CTD
Identified biochemically




activity: a CTD-kinase



Based on its ability to protect RNAPII aginst arrest in a Drosophila tr.system
structure: Heterodimer = 124 kDa + 43 kDa
Cdk9 (også kalt PITALRE) + cyclin T1, T2 or K
Kinase-inactive form without effect on elongation
P-TEFb is inhibited by the nucleotide analogue DRB
Odd S. Gabrielsen
MBV4230
P-TEFb action - release from DSIF/
NELF induced pause

Mechanism of action

Ser 2 phosphorylation of CTD with P-TEFb blocks binding of the elongation -inhibitors NELF and DSIF
(DRB-sensitivity inducing factor)
 Stop and wait for capping: DSIF interacts with hypophosphorylated CTD. NELF recognizes the RNAP
II–DSIF complex and halts elongation. This pause allows the recruitment of the capping enzyme by the
CTD and DSIF (Spt5 subunit), which adds a 5´-cap to the nascent transcript.
Odd S. Gabrielsen
MBV4230
The first steps in elongation






The kinase action of TFIIH phosphorylates Ser5 of CTD
DSIF and NELF followed by the capping machinery (CE) are
recruited into the stalled transcription complex
CE caps the nascent mRNA (see later)
SCPs (small CTD phosphatases) dephosphorylate Ser5.
P-TEFb phosphorylates Ser2 of CTD + the SPT5 subunit of DSIF,
which may facilitate the release NELF.
Trx elongation is resumed through the association of elongation
factors (EFs).
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MBV4230
Elongation - phosphorylation cycle
Odd S. Gabrielsen
MBV4230
HIV and P-TEFb
P-TEFb
Cdk9
Cyclin T
Human specific
5´-
Tat
TAR
CTD-kinase
CTD
RNAPII
Stimulated elongation
Odd S. Gabrielsen
MBV4230
HIV and P-TEFb

HIV-1 produces its own”elongation factor” Tat




Tat is a sequence-specific RNA-binding protein encoded by HIV
Tat binds to a sequence-element TAR (transactivation response element) in the 5´-end
of HIV-transcripts
Tat+TAR promote effective elongation of HIV transcripts
P-TEFb + CTD is required for Tat-function



Ternary complex formed with human cyclin T1+ Tat + TAR (human, not murine T1)
Murine cells become HIV-infectable after transfection with human cyclin T1
Mechanism: Tat facilitates HIV expression by recruiting P-TEFb to TAR, which
improves specifically elongation of HIV-transcripts
Odd S. Gabrielsen
Elongation factors
Helping arrested RNAPII
MBV4230
Pausing and arrest



RNAPII encounters obstacles
during elongation leading to
pausing or arrest
This stage of trx is subject to
control and several genes may
be regulated also on the level of
elongation
Pausing and arrest



result from aberrant backward movement of
RNAPII, leading to displacement of the 3´-OH
end of the growing RNA from the catalytic site
Pausing = reversible
Arrest = not reversible
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MBV4230
Elongation factors that suppress
RNAPII arrest : TFIIS




structure: monomer = 38 kDa
TFIIS binds arrested RNAPII and
TFIIS strongly enhances a weak intrinsic nuclease
activity of RNAPII
TFIIS induces the polymerase to cleave its
nascent transcript, repositioning the new RNA
3´-end within the polymerase catalytic center
Odd S. Gabrielsen
MBV4230
TFIIS helps RNAPII to recover from an
arrested state & resume elongation

Arrest and resuce
 When RNAPII
approaches an arrest site,
it may stop, reverse
direction (backtracking),
and extrude RNA,
leading to transcriptional
arrest.
 TFIIS can rescue arrested
Pol II by inducing
cleavage of the extruded
RNA fragment.
Transcription is then
resumed and continued
past the arrest site.
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RNAPII active site switches from
polymerizing to cleavage

RNAPII contains a single active site for
both RNA polymerization and cleavage
polymerizing
TFIIS induced
cleavage
Odd S. Gabrielsen
Elongation factors
Helping paused RNAPII
MBV4230
Pausing and arrest

Pausing = rate-limiting
step during elongation

RNAPII susceptible to pausing at each
step
 RNAPII cycles between active and
inactive (paused) conformations
Odd S. Gabrielsen
MBV4230
Elongation factors affecting pausing
or arrest

Pausing = ratelimiting step during
elongation

RNAPII susceptible to pausing
at each step
 RNAPII cycles between active
and inactive (paused)
conformations


Pausing = reversible
Arrest = not reversible
Odd S. Gabrielsen
MBV4230
Elongation factors that suppress pausing:
TFIIF, Elongin (SIII), and ELL

TFIIF

Protects the elongation complex against pausing. Acts probably by a
direct but transient interaction with the elongating RNAPII
 phosphorylation of RAP74 stimulates elongation

Elongin




Heterotrimer of subunits A, B and C where A is active, B and C regulatory
Elongins activity can probably be regulated by the von-Hippel-Lindau (VHL)
tumor supressor protein which binds Elongin BC and blocks their binding to
Elongin A.
Genetic disease VHL dispose for several cancers, where mutated VHL binds
Elongin BC less avidly
ELL

80 kDa elongation factor found fused with MLL (mixed lineage leukemia) in
certain leukemias with translocation between chromosome 11 and 19 (ELevennineteen Leukemia)
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Mechanims of action

Pausing = rate-limiting step during elongation

RNAPII susceptible to pausing at each step
 RNAPII cycles between active and inactive (paused) conformations

Elongation factors that suppress pausing,
probably act by decreasing the fraction of time
RNAPII spends in an inactive paused
conformation

For many factors supressing pausing and increasing rate of trx, our
understanding of mechanism is incomplete
Odd S. Gabrielsen
MBV4230
Summary so far
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Odd S. Gabrielsen
Elongation factors
helping RNAPII
through chromatin
Chromatin is an obstacle for the
elongating RNAPII
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Through arrays of nucleosomes propagation of chromatin disruption

Nucleosome arrays
more difficult to pass

Inter-nucleosome contacts
repress elongation
 Induce pausing
 Some elongation factors
stimulate elongation on free
DNA in vitro, but cannot
overcome the chromatin block

In vivo cellular factors
helps to disrupt the
chromatin block to
elongation
Odd S. Gabrielsen
MBV4230
Elongation factors
acting on chromatin

Factors that facilitate elongation through chromatin

SWI/SNF-type chromatin remodellering through ATPdependent mechanisms



Proteins that acetylate (e.g. Gcn5 and Elp3) or methylate histones
FACT - ”facilitates chromatin transcription”- can bind to and destabilize nucleosomes





Swi-Snf and Chd1 remodel nucleosomes
a heterodimer where SPT16 encodes the large subunit
HMG1-like factor SSRP1
Proposed that FACT transiently binds and removes H2A+H2B
Spt4+Spt5 (DSIF) and SPT6 proteins
Reassembly of chromatin after passage of RNAPII
important


To suppress trx initiation from cryptic initiation site (noise)
FACT and SPT6 probably acts by enabling chromatin structure to be disrupted and
then reestablished during trx
Odd S. Gabrielsen
MBV4230
The targeting problem again

How are these factors
targeted to the transcribed
regions of the genome?

Hitching a ride on the RNAPII
Likely through recognizing
hyperphosphorylated CTD



P/CAF (HAT) binds specifically to the
hyperphosphorylated RNAPII
An ”elongator” isolated in yeast that
associates only with the
hyperphosphorylated elongating form of
RNAPII

One of the subunits, Elp3 = HAT
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FACT facilitates chromatin transcription




FACT is a chromatin-specific
elongation factor required for
transcription of chromatin
templates in vitro.
FACT specifically interacts with
nucleosomes and histone H2A/
H2B dimers
FACT appears to act as a
histone chaperone to promote
H2A/H2B dimer dissociation
from the nucleosome and allow
RNAPII transcription on
chromatin
Trx correlates with the
generation of a nucleosome
depleted for one H2A/H2B
dimer
Odd S. Gabrielsen
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FACT

FACT functions to destabilize
the nucleosome by selectively
removing one H2A/H2B dimer,
thereby allowing RNAP II to
traverse a nucleosome.
Odd S. Gabrielsen
MBV4230
The ebb and flow
of histones

The histone chaperone activity of Spt6
helps to redeposit histones on the DNA,


FACT enables the displacement of the
H2A/H2B dimer from the nucleosome,
leaving a “hexasome” on the DNA.


thus resetting chromatin structure after passage of the large
RNAPII complex.
The histone chaperone activity of FACT might help to
redeposit the dimer after passage of RNAPII, thus resetting
chromatin structure.
A possible relationship between histone
acetylation and transcription through
the nucleosome.

In this scenario, HATs associated with RNAPII acetylate the
histone that is being traversed, facilitating its disruption and
displacement.
 Upon redeposition of the displaced histone dimer or
octamer, HDACs immediately deacetylate the histones,
resetting chromatin structure.
Odd S. Gabrielsen
MBV4230
Pattern of histone modifications on
active genes

Methylation and elongation
36
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Histone
Lys methylation

PIC assembly


Ser-5
Promoter clearance


Upstream and downstream of
the PIC, nucleosomes are
dimethylated on H3-K4 and
not methylated at H3-K36.
CTD-kinase of TFIIH
phosphorylates ser-5 of the
CTD resulting in
disengagement from the
promoter and recruitment of
the Set1 complex (HKMT)
and the capping machinery.
Ser-2
Elongation

CTK1 kinase complex (or PTEFb) is recruited to the trx
apparatus resulting in
phosphorylation of ser-2 of
the CTD.
Odd S. Gabrielsen
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HKMT
(SET1)
HKMT
(SET2)
Histone
methylation:
RNAPII dynamic
process
Odd S. Gabrielsen
MBV4230

In yeast two separate HKMTcontaining complexes associate with
RNAP II and are implicated in histone
methylation at mRNA coding regions



tri-methylation of H3-K4 catalyzed by
Set1 accumulate near the 5´-mRNA
coding region of genes


Set1 is implicated in establishing H3-K4 histone
methylation.
Set2 is implicated in establishing H3-K36 histone
methylation.
and is associated with the early stages of transcription.
Set2 specifically associates with the
elongating form of RNAP II.

Set2-mediated H3-K36 methylation, along with di-methyl
H3-K4, corresponds to later stages of elongation.
Odd S. Gabrielsen
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PAF complex

The yeast Set1 and Set2 HKMTs are recruited by the PAF
trx elongation complex in a manner dependent upon the
phosphorylation state of the CTD of RNAPII


The PAF complex directly recruits Set1 to the trx machinery by bridging the
interaction between RNAP II and Set1
PAF has five subunits


Paf1, Rtf1, Cdc73, Leo1, and Ctr9
Evidence suggests that PAF integrates transcriptional regulatory signals and
coordinates modifications affecting chromatin
Odd S. Gabrielsen
MBV4230
A possible logic?
Ass factors
Ass factors


The CTD of RNAPII has been found to anchor several
proteins with a role in elongation and pre-mRNA
processing
A histone code of methylated histone-tails may provide
additional anchorage sites for elongation factors or
processing enzymes
Odd S. Gabrielsen
Pre-mRNA processing
Processes tightly linked to
elongation
MBV4230
A role for CTD in mRNA processing?

Several novel CTD-binding proteins
identified the last few years with functions
in splicing and termination

Tight coupling : transcription - pre-mRNA
processing
Pre-mRNA
(hnRNA)
AAAAAAAAAAAAA
ca
mRNA
Odd S. Gabrielsen
MBV4230
CTD-mediated coupling :
transcription - pre-mRNA processing

Pre-mRNA processing

Capping
 Splicing
 Cleavage/polyadenylation

Physical contact between the machines for for
transcription and pre-mRNA processing
through CTD
Odd S. Gabrielsen
Capping
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Cotranscriptional ”Capping”


Pre-mRNA modified with 7methyl-guanosine triphosphate
(cap) when RNA is only 25-30
bases long
Cap: 3 modifications

7-met-guanosine coupled to 5´-end



O2´-methylation of ribose



Coupling by 5´-5´triphosphate bridge
Takes place co-transcriptionally
Cap2, Cap1 (multicellulær), Cap0 (unicellulær)
N6-methylation of adenine
Capping occurs cotranscriptionally
Cap1
Cap2
Odd S. Gabrielsen
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Capping

3 enzymes
involved

1. RNA 5´-Triphosphatase
(RTP) removes a
phosphate
 2. Guanylyl transferase
(GT) attach GMP


Enzyme 1+2 coupled: in
multicellular organisms:
in same polypeptid, in
yeast heterodimers
3. 7-methyltransferase
(MT) modifies the
terminal guanosine
2
.
1
.
3
.
Odd S. Gabrielsen
MBV4230
Cotranscriptional ”Capping”

CTD recruits capping enzyme as soon as it is
phosphorylated





CTD required for effective capping
Guanylyl transferase (mammalian + yeast) binds directly to phosphorylated CTD,
not to non-phosphorylated
7-methyltransferase (yeast) binds also phosphorylated CTD
phosphorylated CTD may also regulate the activity of
the enzymes
Cap structure is recognized by CBC (Cap binding
complex)

Composed of two proteins CBP20 and CBP80

Major role in stabilization, block exonucleases
 CBC stimulates subsequent splicing and 3´-end processing
Odd S. Gabrielsen
Splicing
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Splicing

Splicing of introns occurs cotranscriptionally

EM evidence
 Half-life BR1 intron only 2.5 min ≈ 5 kbs elongation of RNAPII

Splicing depends on CTD

Inhibited by CTD truncation
 In vitro splicing stimulated by added phosphorylated CTD

CTD binds probably splicing-factors

Not fully characterized

CTD associated with SR- and Sm-splicing factors
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Splicing - excision of lariat
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Cotranscriptional
splicing
Odd S. Gabrielsen
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Association CTD-splicing factors

CTD binds probably splicing-factors



CTD associated with SR- and Sm-splicing factors
CASP (CTD-associated SR-like proteins) and SCAF (SR-like CTD-associated
factors)
RNA-binding proteins due to



Promoter-context can determine associated SR
proteins and hence splicing



RRM-domains target the factor to exon enhancer sequences
RS-domains acting as ”glue” by forming RS-RS interactions
Fibronectin: one intron included or excluded depending on the promoter
Model: SR-CTD interaction set up during intiation, thus priming the elongation
complex
Elongation rate can determine choice of alternative
splice sites
Odd S. Gabrielsen
3´-end formation
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Modification of 3´- end:
poly-adenylation

Defined 3´-end is formed not by
precise termination, but as a result of
processing

Pre-mRNA heterogenous 3´-ends,
 mRNA well defined 3´-ends

Poly(A) tails added in 3´-end

Ca 200x adenosines in a stretch of As added in a
particular process

I.e. poly(A) not gene encoded
AAAAAAAAAAAAA
cap
Odd S. Gabrielsen
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Trimming of 3´-end
cap
Inprecise
termination
0 0
AAAAAAAAAAAAA
cap
Precise end after
cleavage and polyadenylation
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Poly-adenylation
- two-step process

1.cleavage 15-25 downstream of AAUAAA




within 50 nt before a less conserved (G)U-rich element (DSE)
cleavage preferentially in a CA nucleotide
2. Poly(A) tail made by a poly(A) polymerase
Recognition:

AAUAAA binds CPSF through its largest subunit (of four in total)




Cleavage and polyadenylation specificity factor
DSE binds Cleavage stimulatory factor CstF
In addition two other ”cleavage factors” CF-I and -II
Coupled processes:


CPSF and CstF stimulates each other
bound CPSF stimulates the poly(A) polymerase
Odd S. Gabrielsen
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Cleavage and polyadenylation

6 multimeric protein
factors involved






PAP (poly (A) polymerase)
PABP II (poly(A)-binding protein)
CPSF
CstF
CF-I
CF-II
Odd S. Gabrielsen
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Processing of 3´-end: ”Cleavage/
polyadenylation”

When RNAPII is approaching the 3´-end of the
transcript, several coupled processes are taking place






Splicing of terminal intron
cleavage at poly(A)-site,
addition of poly(A) tail,
termination downstream of poly(A)-site and liberation of RNAPII
These av difficult to separate in time
These processes depend on CTD




Splicing, processing of 3´-end and termination downstream of poly(A) site are all
inhibited by CTD truncations
”Cleavage-polyadenylation specificity factor” CPSF and ”cleavage stimulation
factor” CstF bind specifically to CTD and are found associated with holoRNAPII.
Poly(A) polymerase is NOT associated with RNAPII
CPSF is TBP-associated - becomes at some stage transferred from TFIID to CTD
Odd S. Gabrielsen
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Molecular interactions between
mRNA processing reactions

Several steps stimulates other steps in the process


Eks 1: Cap stimulates splicing of first intron
Eks 2: Cap stimulates 3´-end cleavage (but not polyadenylation)
Odd S. Gabrielsen
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Models for trx termination - A

The allosteric model (A)

During elongation, RNAPII is in a highly
processive conformation (green oval).

RNAPII is transformed into a
nonprocessive form (red octagon) after
transcribing through the poly(A) site
(AATAAA).

The RNA transcript

red upstream of and

blue downstream of

the poly(A) cleavage site (lightening bolt).

Dotted blue line = degraded RNA.

5´cap, added cotrx, = pale blue hat
Odd S. Gabrielsen
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Models for trx termination - B

The torpedo model (B)


RNA downstream of the poly
(A) cleavage site (blue line) is
digested by a 5´-3´exonuclease
(Rat1 in yeast and hXrn2 in
humans (blue pacman), which
tracks with RNAPII throughout
the length of the gene.
After poly(A) site cleavage, the
exonuclease torpedo is guided
along the RNA to its
polymerase target and
dissociates it from the DNA
template.
Odd S. Gabrielsen
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A combined model

where the exonuclease cooperates with an unknown helicase
and/or allosteric modulator of the polymerase, converting it from
processive to nonprocessive form, ultimately disrupting the
RNA-DNA hybrid and releasing the polymerase.
Odd S. Gabrielsen
MBV4230
Cotranscriptional
processing
RNAPII = mRNA factory
that is orchestrating a
coupled series of events
including transcription,
capping, splicing and
processing of 3´-end
Odd S. Gabrielsen