LETTER
doi:10.1038/nature09785
Structural basis of RNA polymerase II backtracking,
arrest and reactivation
Alan C. M. Cheung1 & Patrick Cramer1
During gene transcription, RNA polymerase (Pol) II moves
forwards along DNA and synthesizes messenger RNA. However,
at certain DNA sequences, Pol II moves backwards, and such backtracking can arrest transcription. Arrested Pol II is reactivated by
transcription factor IIS (TFIIS), which induces RNA cleavage that is
required for cell viability1. Pol II arrest and reactivation are involved
in transcription through nucleosomes2,3 and in promoter-proximal
gene regulation4–6. Here we present X-ray structures at 3.3 Å resolution of an arrested Saccharomyces cerevisiae Pol II complex with
DNA and RNA, and of a reactivation intermediate that additionally
contains TFIIS. In the arrested complex, eight nucleotides of backtracked RNA bind a conserved ‘backtrack site’ in the Pol II pore and
funnel, trapping the active centre trigger loop and inhibiting
mRNA elongation. In the reactivation intermediate, TFIIS locks
the trigger loop away from backtracked RNA, displaces RNA from
the backtrack site, and complements the polymerase active site with
a basic and two acidic residues that may catalyse proton transfers
during RNA cleavage. The active site is demarcated from the backtrack site by a ‘gating tyrosine’ residue that probably delimits backtracking. These results establish the structural basis of Pol II
backtracking, arrest and reactivation, and provide a framework
for analysing gene regulation during transcription elongation.
Backtracking of bacterial RNA polymerase and the related eukaryotic
Pol II to an arrested state is triggered by a weak DNA–RNA hybrid, and
dislodges the RNA 39 end from the active site7–9. A recent study
attempted to crystallize an arrested Pol II complex with the use of
DNA–RNA scaffolds containing 39-overhanging RNA10. This allowed
the visualization of one or two backtracked RNA nucleotides, but not
further-backtracked RNA10. We have now resolved further-backtracked
RNA nucleotides by crystallization of an arrested complex obtained by
Pol II transcription of a 39-tailed DNA template. Tailed template transcription was previously used for structural studies11, and is prone to
arrest12. We incubated Pol II with a tailed template (Fig. 1a) and different
NTP substrates, crystallized the obtained complexes, collected diffraction data, and inspected difference electron density maps after phasing
with the free Pol II structure. Only incubation with CTP alone resulted
in interpretable difference density for backtracked RNA (Fig. 1b). The
register of the nucleic acids was defined by bromine labelling of the 25
template base (Fig. 1a, b) and the structure was refined at 3.3 Å resolution (Methods and Supplementary Table 1).
The structure revealed 13 base pairs (bp) of downstream DNA, a
6-bp hybrid, and 9 nucleotides of single-stranded 39 RNA that is
extruded through the pore into the funnel (Fig. 1). Observation of a
15-nucleotide RNA with 9 unpaired residues is consistent with arrest
of tailed template transcription after synthesis of 13–17 nucleotides12,
with a binding site on Pol II for a 9-nucleotide 39 RNA13, and with
patterns of RNA cleavage and nuclease protection in arrested complexes14,15. Thus, the template had allowed for CMP mis-incorporation,
and the resulting destabilized hybrid drove backtracking to the arrested
state.
Comparison with the elongation complex structure reveals that the
DNA–RNA hybrid is tilted towards the bridge helix (Fig. 1c). In the
1
elongation complex, the RNA 39 end occupies position 21, and the
incoming NTP substrate and the templating DNA base occupy position 11, relative to the active site. In the arrested complex, the 21 base
pair is tilted by ,25u. Its DNA base resides in the site that is normally
occupied by the 11 nucleotide, which instead resides in the downstream cleft, leaving the non-complementary 11 RNA base unpaired.
In contrast, the 11 RNA base is paired with the DNA template base in
a previously reported complex backtracked by one position10. Whether
hybrid tilting is a cause or consequence of backtracking or whether it
results from the shorter hybrid remains to be investigated.
The structure shows that backtracked RNA is extruded into the pore
and funnel (Fig. 1d), confirming an early hypothesis16. The key feature
of the complex is the highly defined structure of backtracked RNA and
its extended binding site (Fig. 2). Backtracked RNA binds a ‘backtrack
site’ along one side of the pore and the mobile trigger loop on the
opposite side. The trigger loop has been proposed to control the lateral
oscillation of polymerase17. Its ‘trapped’ conformation observed here is
distinct from the closed, open and wedged conformations that occur
during forward elongation18–21 (Supplementary Fig. 1), and also different from the conformation observed in a complex backtracked by
one nucleotide10. These observations indicate the basis for transcription arrest. When backtracking is not extensive, RNA interactions with
the backtrack site are partial and weak, and Pol II can spontaneously
move forward and continue elongation. When backtracking is more
extensive, backtracked RNA and the trigger loop are trapped, preventing forward elongation and arresting Pol II.
The structure reveals details of backtracked RNA and its interactions with the backtrack site (Fig. 2 and Supplementary Table 2). The
first backtracked RNA nucleotide 12 contacts the bridge helix residue
T827 and the fork residue E529, and stacks between the 11 base and
Rpb2 residue Y769 that we call the ‘gating tyrosine’. The 12 base also
contacts the gating tyrosine in a previous structure, although via an
edge-to-face interaction10. Beyond position 12, base stacking discontinues due to steric hindrance by the gating tyrosine. The RNA
backbone kinks between nucleotides 12 and 13, and contacts Rpb2
residue R766. The 13 nucleotide binds the trigger loop residues Q1078
and T1080, consistent with crosslinking data22. Beyond position 13,
RNA binds exclusively to the Rpb1 funnel domain. A stack of bases
from nucleotides 13 to 15 is followed by nucleotide 16, which inserts
its base into a pocket that is flanked by R726 and I756 and called here
the ‘funnel pocket’. Between positions 16 and 17, the RNA kinks
again. The 17 base stacks against residue F755 on one side and against
the 18 and 19 bases on the other. Because RNA-binding residues are
conserved among eukaryotes (Supplementary Table 2 and Supplementary Fig. 2), backtracked RNA probably binds the same backtrack site
in all Pol II enzymes. Modelling purine bases onto the backtracked
RNA reveals potential clashes with Pol II (Supplementary Fig. 3). This
indicates that the backtrack site preferentially accommodates pyrimidine bases, and provides an explanation for why known DNA arrest
sites direct synthesis of pyrimidine-rich RNA23.
The RNA conformation and Pol II interactions indicate why intrinsic
RNA cleavage generally occurs after backtracking by one position, a
Gene Center and Department of Biochemistry, Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universität München, Feodor-Lynen-Str. 25, 81377 Munich, Germany.
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RESEARCH LETTER
a
DNA–RNA
G
hybrid
Downstream DNA
d
A G G T A A G C T A G C T
C A A T C T C G T B G T G G T C C A T T C G A T C G A
Single-stranded C C C C C C C C C C C C C C C 3′
template
–5
Active site
Backtracked
+9 RNA
–2 +1
RNA synthesized in situ
5-Bromouracil
Mg A
Non-template DNA
Template DNA
b
Downstream DNA
Br
Mg A
Bridge helix
Side view
RNA backtrack
site
90°
Backtracked RNA
c
Pol II
Bridge helix
Trigger loop
Fork loop
Hybrid binding domain
Funnel domain
Active site
Rpb 4/7
Template DNA (arrested)
RNA (arrested)
Elongation complex
~25° tilt
of –1 bp
–1
Mg A
–2
–1
–1
–1
+1
+2 +3
+1
+1
Backtracked RNA
+2
Gating
tyrosine
Front view
Funnel and
pore
Front view
Figure 1 | Structure of arrested Pol II. a, Schematic of nucleic acids. Ordered
nucleotides are shown with filled circles. The colour code is used throughout.
The NTP-binding site corresponds to position 11 and backtracked
(downstream) residues are labelled with positive numbers. Some complexes
may contain additional disordered nucleotides at the RNA 39 end. b, Unbiased
difference electron density (blue mesh, contoured at 3.0s) for nucleic acids,
after phasing with the Pol II structure. A bromine atom is revealed by
anomalous difference density (magenta mesh, contoured at 4s, peak height
7s) and defined the nucleic acid register. c, Front view16 of a superposition of
the tilted DNA–RNA hybrid in the arrested Pol II structure with that in the
structure of the elongation complex (light green). d, Side and front views of the
arrested Pol II complex structure with functional elements highlighted.
phenomenon that underlies mRNA proofreading10,24,25. Backtracking
begins with Pol II pausing and fraying of the 39-terminal RNA nucleotide 11 against the gating tyrosine24,26. The gating tyrosine maintains
contact with the 39 nucleotide during the first step of backtracking10
(Fig. 2a). Backtracking by one position may thus be facilitated, but
further backtracking is probably disfavoured because RNA base stacking must be discontinued at the gating tyrosine. Hence, the gating
tyrosine may generally delimit the extent of backtracking. If, however,
base-stacking interactions and the hybrid are weak, backtracking
beyond the gating tyrosine may occur and lead to arrest.
Arrested Pol II is reactivated by TFIIS, which induces cleavage of
backtracked RNA. TFIIS binds with its domain II near the rim of the
Pol II funnel, and extends into the Pol II pore with its domain III, which
reaches the active site with a b-stranded hairpin19,27. Superposition of
the arrested complex with the Pol II–TFIIS complex27 revealed that the
backtracked RNA overlaps with TFIIS domain III. This indicated that
backtracked RNA prevents TFIIS from invading the pore, posing the
question of how reactivation occurs. To investigate this, we soaked
arrested complex crystals with a TFIIS variant that carries two point
mutations in functionally essential acidic hairpin residues (D290A/
E291A), and solved the structure of the resulting reactivation intermediate at 3.3 Å resolution (Methods). As observed before19, TFIIS
changed the Pol II conformation, realigned RNA in the hybrid,
inserted its domain III into the pore, and reached the active site with
its hairpin.
In the structure of the reactivation intermediate, TFIIS binding
moved the trigger loop by up to 5 Å from the trapped to the ‘locked’
position (Fig. 3a) observed previously27. TFIIS binding also induces a
rotation of the gating tyrosine side chain, which prevents its stacking
with backtracked RNA. Backtracked RNA was displaced from the
backtrack site into the part of the pore that remains accessible after
binding of TFIIS domain III (‘restricted pore’, Fig. 3b). The electron
density for displaced RNA was discontinuous and only allowed for
backbone modelling (Fig. 3b). The discontinuous density apparently
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LETTER RESEARCH
a
Gating
tyrosine
Y769
Mg A
+2
Bridge helix
Mg A
+1
Rpb1 T827
Rpb2 R766
Q1078
R766
T827
Rpb1
Q1078 +3
T1080
Q1078
Backtracked RNA
+3
K752
+4
+5 T1080
I756
+6
Mg A •
+1
+1
+2
b
E529
Trapped
trigger
loop
90°
S754
K752
+5
I756
+3
Rpb2
+2
E529
Rpb2 Y769
Rpb1 K752
+4
+4
Rpb1 S754
+5
T1080
Rpb1 I756
+6
+6
R726
R726
F755
F755
Rpb1 R726
+5
+7
Rpb1 S754
Rpb1 R731
+6
+8
+9
R731
Rpb1 F755
+7
I756
+8
R726
+9
R731
Side view
Front view
Rpb1 funnel domain
+9
Funnel pocket
Figure 2 | Backtracked RNA in the backtrack site. a, Side and front views of backtracked RNA. RNA-binding Pol II elements in the pore and funnel are depicted,
and contact residues in the backtrack site are labelled. b, Schematic of Pol II interactions with backtracked RNA.
b
a
90°
Locked
trigger loop
Displaced
backtracked
RNA
TFIIS
linker
TFIIS
domain III
TFIIS domain II
Displaced
backtracked
RNA
c
Restricted
pore
Mg A
Mg A
Backtracked RNA in
arrested complex
Side view
Front view
d
e
Scissile bond
–2
–1
mFo–DFc 3σ
Mg A
Displaced
backtracked
RNA
Front view
+1
Phosphate
+1
Backtracked RNA
TFIIS
domain III
TFIIS
linker
R287
TFIIS domain II
W
D290
3′-OH of
RNA in EC
A
B
Rpb1
aspartate
loop
Figure 3 | Structure of reactivation intermediate. a, Side view. TFIIS
domains II and III are in green and orange, respectively, and the domain II–III
linker is in yellow. b, Displacement of backtracked RNA from the backtrack site
into the restricted pore. Difference electron density for displaced backtracked
RNA is contoured at 2s (blue) and 2.5s (red). On the right, backtracked RNA
from the arrested complex is modelled into the structure, revealing a clash with
TFIIS domain III. c, Unbiased difference electron density for TFIIS after
phasing with the Pol II structure contoured at 3s, and sigmaA-weighted by
E291
TFIIS hairpin
Rpb2 D837
Mg A
Elongation complex (EC)
coefficients m and D. d, Model for active site geometry during TFIIS-induced
RNA cleavage. Metal A is coordinated by the Pol II aspartate loop and the RNA
11 phosphate. Metal B, the nucleophilic water molecule (W, blue sphere), and
side chains of TFIIS hairpin residues D290 and E291 (orange) are placed onto
the crystal structure without clashes. This supports a catalytically competent
arrangement for an SN2 mechanism. The arrow indicates the direction of the
in-line nucleophilic attack. e, Superposition of the view in d with the active site
conformation of the Pol II elongation complex19.
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RESEARCH LETTER
reflects rotational mobility rather than partial release of backtracked
RNA, as RNA was not cleaved in a reconstituted backtracked complex
by the inactive TFIIS variant (not shown). These observations indicate
that TFIIS weakens the Pol II grip on backtracked RNA, and displaces
and mobilizes RNA by competitive binding to the backtrack site.
Pol II reactivation by TFIIS-dependent cleavage apparently involves
two metal ions and a nucleophilic water molecule27–29. Metal A is persistently bound to the active site aspartate loop16, whereas metal B may
be recruited by TFIIS27,29. The reactivation intermediate structure provides new insights into TFIIS-induced cleavage, as it is at higher resolution than previous work10,19, and because it reveals the RNA 11
nucleotide, and thus the scissile phosphodiester bond between nucleotide 21 and 11 (Fig. 3d). First, metal A binds the 11 RNA phosphate
to align the scissile bond, in contrast to its binding of the RNA 39
hydroxyl during nucleotide addition (Fig. 3e). Second, the TFIIS
hairpin residue R287 reaches the catalytic site and could stabilize the
negatively charged transition state, explaining its role in catalysis30.
Third, modelling the side chains of D290 and E291 onto the structure
Strong
hybrid
TSS
Weak
hybrid
Initiation
METHODS SUMMARY
Pol II
Active site
Mg A
indicates that they can coordinate metal B, together with the Rpb2
residue D837 (Fig. 3d). The invariant charged TFIIS residues R287,
D290 and E291 may be required for two catalytic proton transfers,
proton subtraction from the nucleophilic water, and proton donation
to the product RNA 39 terminus.
The presented structural snapshots of transcription intermediates
reveal the mechanisms of Pol II backtracking, arrest and reactivation
(Fig. 4). When Pol II encounters a DNA sequence that impairs elongation, it pauses and backtracks by one position. Because further backtracking is hindered by the gating tyrosine, polymerase-intrinsic
cleavage of a dinucleotide from the RNA 39 end can occur, and elongation continues. However, at an arrest site the hybrid is weak and the
RNA can backtrack beyond the gating tyrosine. Extensive backtracking traps RNA and the trigger loop in the pore, inhibiting elongation
and arresting Pol II. TFIIS reactivates arrested Pol II by locking the
trigger loop away from RNA, displacing and mobilizing backtracked
RNA in the pore, and complementing the active site with a basic and
two acidic side chains. This induces cleavage and release of backtracked RNA, and creates a new RNA 39 end at the active site that
allows transcription to resume.
Downstream DNA
Pore
Funnel
domain
Pol II elongation
complex
Pausing
Saccharomyces cerevisiae 12-subunit Pol II was prepared as described24. Purified
Pol II (3.5 mg ml21) was mixed with a twofold molar excess of tailed template
(Fig. 1a) prepared as described19, 8 mM magnesium chloride and 2 mM CTP, and
incubated for 2 h at 20 uC before crystallization by vapour diffusion with 6% PEG
6000, 200 mM ammonium acetate, 300 mM sodium acetate, 50 mM HEPES
pH 7.0 and 5 mM TCEP as reservoir solution. Crystals were grown for 5–10 days,
cryo-protected in mother solution supplemented with 22% glycerol and containing 2 mM CTP, 8 mM magnesium chloride, and 4 mM tailed template, followed by
overnight incubation at 8 uC before harvesting and freezing in liquid nitrogen.
Arrested Pol II–TFIIS complex crystals were prepared by adding the inactive TFIIS
variant D290A/E291A27 to the cryo-protectant at 1 mg ml21 and incubating
arrested Pol II crystals overnight at 8 uC. Diffraction data at 3.3 Å were collected
at 100 K at beamline X06SA of the Swiss Light Source and structures were solved
with molecular replacement using the model of 12-subunit Pol II (1WCM). Data
were collected at 13.494 keV, the K-absorption peak of bromine.
Backtracking
Received 13 October; accepted 23 December 2010.
Published online 23 February 2011.
Gating tyrosine
Further
backtracking
and arrest
1.
2.
Trapped trigger loop
3.
4.
Backtracked RNA in
backtrack site
TFIIS binding
and RNA
displacement
Locked trigger loop
5.
6.
7.
Displaced backtracked
RNA in restricted pore
8.
9.
RNA cleavage
and release
10.
11.
Released
RNA fragment
Figure 4 | Mechanism of Pol II backtracking, arrest and reactivation.
Schematic of Pol II functional states. For details, compare text.
12.
13.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank F. Brueckner, G. Damsma, K. Kinkelin, D. Kostrewa,
L. Larivière, E. Lehmann, F. Martinez, S. Sainsbury and J. Sydow. Part of this work was
performed at the Swiss Light Source at the Paul Scherrer Institut, Villigen, Switzerland.
P.C. was supported by the Deutsche Forschungsgemeinschaft, SFB646, TR5,
FOR1068, NIM, Bioimaging Network BIN, and the Jung-Stiftung.
Author Contributions A.C.M.C. carried out experiments. P.C. supervised the project.
A.C.M.C. and P.C. prepared the manuscript.
Author Information Coordinates and structure factors of the arrested Pol II elongation
complex and the arrested Pol II reactivation intermediate have been deposited with the
Protein Data Bank under accession numbers 3PO2 and 3PO3, respectively. Reprints
and permissions information is available at www.nature.com/reprints. The authors
declare no competing financial interests. Readers are welcome to comment on the
online version of this article at www.nature.com/nature. Correspondence and requests
for materials should be addressed to P.C. ([email protected]).
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