Elongation and

pre-mRNA processing

RNAPII

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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

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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--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 23-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.

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Stage 1 - the early steps:

from ITC to EEC

6
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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.
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Several elongation-factors isolated that

control pausing or stimulate elongation
-

P-TEFb
TFIIS

NELF
DSIF

TFIIF
Elongin
FACT

RNAPII
+

ELL

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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 IIDSIF 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.

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Elongation stage 3
Productive elongation

11

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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 (ogs kalt PITALRE) + cyclin T1, T2 or K
Kinase-inactive form without effect on elongation

P-TEFb is inhibited by the nucleotide analogue DRB

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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
IIDSIF 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.

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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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Elongation - phosphorylation cycle

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HIV and P-TEFb

P-TEFb
Cdk9

Cyclin T
Human specific

5-

Tat
TAR

CTD-kinase

CTD

RNAPII
Stimulated elongation

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HIV and P-TEFb

HIV-1 produces its ownelongation 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

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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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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

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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

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Elongation factors
Helping paused RNAPII

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Pausing and arrest

Pausing = rate-limiting
step during elongation

RNAPII susceptible to pausing at each

step
RNAPII cycles between active and
inactive (paused) conformations

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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

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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

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Summary so far

28
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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
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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

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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
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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.

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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.

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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.
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HKMT
(SET1)

HKMT
(SET2)

Histone
methylation:
RNAPII dynamic
process

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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.

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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
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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
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Pre-mRNA processing
Processes tightly linked to
elongation

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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

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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

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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 5-5triphosphate bridge

Takes place co-transcriptionally
Cap2, Cap1 (multicellulr), Cap0 (unicellulr)

N6-methylation of adenine

Capping occurs cotranscriptionally

Cap1
Cap2

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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
.

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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

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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

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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

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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
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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

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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

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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

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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)

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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.

5cap, added cotrx, = pale blue hat

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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 5-3exonuclease
(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.

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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.

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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