Architecture of initiation-competent 12-subunit
RNA polymerase II
Karim-Jean Armache, Hubert Kettenberger, and Patrick Cramer*
Institute of Biochemistry and Gene Center, University of Munich, Feodor-Lynen-Strasse 25, 81377 Munich, Germany
Edited by E. Peter Geiduschek, University of California at San Diego, La Jolla, CA, and approved March 27, 2003 (received for review January 31, 2003)
RNA polymerase (Pol) II consists of a 10-polypeptide catalytic core
and the two-subunit Rpb4?7 complex that is required for tran-
scription initiation. Previous structures of the Pol II core revealed a
‘‘clamp,’’ which binds the DNA template strand via three ‘‘switch
regions,’’ and a flexible ‘‘linker’’ to the C-terminal repeat domain
(CTD). Here we derived a model of the complete Pol II by fitting
structures of the core and Rpb4?7 to a 4.2-Å crystallographic
electron density map. Rpb4?7 protrudes from the polymerase
DNA loading. Rpb7 forms a wedge between the clamp and the
linker, restricting the clamp to a closed position. The wedge
allosterically prevents entry of the promoter DNA duplex into the
active center cleft and induces in two switch regions a conforma-
tion poised for template-strand binding. Interaction of Rpb4?7
with the linker explains Rpb4-mediated recruitment of the CTD
phosphatase to the CTD during Pol II recycling. The core–Rpb7
interaction and some functions of Rpb4?7 are apparently con-
served in all eukaryotic and archaeal RNA polymerases but not in
the bacterial enzyme.
transcription by Pol II underlies cell proliferation and differen-
tiation. Regulation occurs mainly at the level of transcription
initiation, when Pol II assembles with the general transcription
factors TFIIB, -D, -E, -F, and -H into the initiation complex on
promoter DNA (1–3). In addition to the 10-subunit Pol II core,
initiation requires the heterodimeric Rpb4?7 complex that can
dissociate from core (4, 5).
Crystallographic structures are available for the Saccharomyces
cerevisiae Pol II core (6, 7), which is sufficient for RNA elongation
(4, 5). In the core structures, the two large subunits form opposite
sides of a central ‘‘cleft,’’ and the eight small subunits are arrayed
around the periphery. The cleft contains the active center and is
constricted at one end by a protein ‘‘wall.’’ One side of the cleft is
formed by a mobile ‘‘clamp,’’ which adopts open states in two
crystal forms of the Pol II core (6, 7) but is closed in a further
structure of a core elongation complex with bound template DNA
and product RNA (8). Mobility of the clamp relies on five protein
‘‘switch’’ regions, which connect the clamp to the remainder of the
enzyme (7, 8). A flexible ‘‘linker’’ emerges from the core surface
below the clamp and connects to the C-terminal repeat domain
(CTD) of the largest Pol II subunit, which is disordered in all core
Counterparts of the Rpb4?7 complex exist in the two other
eukaryotic nuclear RNA polymerases (9–13) and the archaeal
RNA polymerase (14). The structure of an archaeal Rpb4?7
homolog (15). To understand Rpb4?7 function and elucidate the
mechanism of transcription initiation, we studied the 12-subunit
Pol II by x-ray crystallography.
NA polymerase (Pol) II synthesizes all eukaryotic mRNA in
the course of gene transcription. The regulation of gene
Materials and Methods
The yeast Pol II core was purified from a yeast rpb4 deletion
strain essentially as described (16). Yeast Rpb4 and Rpb7 were
coexpressed in Escherichia coli as described (17), purified to
homogeneity, and concentrated to 5–10 mg?ml. For reconstitu-
tion of stoichiometric 12-subunit Pol II, 0.5 mg of core was
incubated for 1 h at 20°C with a 5-fold excess of Rpb4?7, and
unbound Rpb4?7 was removed by gel filtration (Superose 6,
Amersham Biosciences). Fractions containing the 12-subunit
Pol II were concentrated to 4 mg?ml. Crystals were grown at
20°C with the hanging-drop method by using a reservoir solution
of 8% PEG 20000?360 mM NaH2PO4/(NH4)2HPO4, pH 6.0?50
mM dioxane?5 mM DTT. Crystals grew to a maximum size
of 0.4 ? 0.2 ? 0.1 mm. Crystals were transferred stepwise to
reservoir solutions with 5%, 10%, 18%, and 22% ethylene glycol
or glycerol, incubated at 4°C overnight, mounted in cryoloops,
and dispersed and stored in liquid nitrogen. Diffraction was
observed beyond 4-Å resolution, but radiation damage and
anisotropy limited complete data to 4.2-Å resolution. The high-
est peak in molecular replacement with AMORE (18) was ob-
tained by using as search model the core elongation complex
structure (correlation coefficient ? 45.1?18.4 for first?second
peak, 10–6.5 Å) (8). This structure was used without nucleic
acids as a model for the core. Eight additional residues of the
Rpb1 linker were included. The archaeal Rpb4?7 counterpart
structure was manually fitted to 2Fobs? Fcalcand Fobs? Fcalc
electron density maps phased with the core model. Manual
rigid-body adjustments accounted for slight changes in the
relative position of three domains (Rpb4, yeast Rpb7 residues
1–80, and yeast Rpb7 residues 81–169). Residues 117 and
174–187 of the archaeal Rpb7 counterpart were deleted, and
yeast Rpb7 residues 52, 53, 155, and 156 were inserted. An
insertion between ?-helices H1 and H2 in yeast Rpb4 could not
be modeled. Although an additional helical density was ob-
served, the connectivity was uncertain. No continuous density
was observed for an N-terminal extension of yeast Rpb4. Despite
weak sequence similarity, the overall fold of the C-terminal half
of yeast Rpb4 corresponds to that of its archaeal counterpart and
is confirmed by the location of selenomethionine 145 in yeast
Rpb4, which corresponds to residue 38 in helix 2 of the archaeal
Rpb4 counterpart structure. At the resolution of the data, no
refinement was carried out, and the fitted structures were
reduced to C?backbones. Ordering of the switches was con-
firmed with a simulated annealing omit map, calculated with a
model lacking switches 1–3, with the program CNS (19).
Results and Discussion
Reconstitution and Structural Analysis of Pol II. Endogenous yeast
Pol II contains substoichiometric amounts of Rpb4?7, which
impede crystallization. To overcome this difficulty, yeast Pol II
was reconstituted from endogenous core and recombinant
Rpb4?7 (compare Materials and Methods). The reconstituted
12-subunit polymerase could be purified by size-exclusion chro-
matography and formed large single crystals within several days.
This paper was submitted directly (Track II) to the PNAS office.
Data deposition: The atomic coordinates have been deposited in the Protein Data Bank,
www.rcsb.org (PDB ID code 1NT9).
Abbreviations: Pol, RNA polymerase; CTD, C-terminal repeat domain.
See commentary on page 6893.
*To whom correspondence should be addressed. E-mail: cramer@LMB.uni-muenchen.de.
June 10, 2003 ?
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no. 12 www.pnas.org?cgi?doi?10.1073?pnas.1030608100
With the use of cryocooling and synchrotron radiation, complete
diffraction data to 4.2-Å resolution were obtained (Table 1),
which allowed for structure solution by molecular replacement.
Initial electron density maps were phased with a truncated core
model that lacked the clamp. These maps showed positive
difference density for the clamp and for an additional mass on
the core surface, which could be fitted with the structure of the
archaeal Rpb4?7 counterpart (Fig. 1A; ref. 15). The orientation
of Rpb4?7 was additionally confirmed by labeling Rpb4?7 with
selenomethionine (Fig. 1B). The resulting backbone model
reveals the location of amino acid residues in all 12 Pol II
Overall Structure. The model shows a single Rpb4?7 complex on
the outside of the core protruding from the base of the clamp
(Fig. 2A). The location of Rpb4?7 agrees with that in cryoelec-
tron microscopic reconstructions of Pol II (20). However, the
orientation of Rpb4?7 differs. Whereas electron microscopy
suggested that Rpb4 binds to the Pol II core, our data unam-
biguously show that instead Rpb7 binds to the Pol II core. The
Rpb4?7 location also agrees with that of the Rpb4?7 counterpart
in Pol I, also observed by electron microscopy (11, 21). Most of
the Rpb4?7 surface is exposed and accessible for interactions
with proteins or nucleic acids. Exiting RNA may interact with a
potential nucleic acid-binding surface of Rpb7 (15, 22) that faces
the Pol II ‘‘saddle,’’ from which RNA emerges (Fig. 2B). Such
interactions may slightly change the Rpb4?7 orientation that is
fixed here by crystal packing.
Rpb4?7 binds to the Pol II core with the N-terminal ribonu-
cleoprotein-like domain of Rpb7 (15), termed here the ‘‘tip’’
(Fig. 2A). The remainder of Rpb4?7 extends far from the core
surface, explaining why mutations in this region do not impair
core binding (22). Consistent with the core–Rpb7 interaction,
Rpb7 alone can bind to core (23), and Rpb7 is essential for yeast
growth (24), whereas Rpb4 is not (25). Deletion of the rpb4 gene
in yeast facilitates dissociation of Rpb7 from core (5). Our model
suggests that loss of the Rpb4–Rpb7 interface after Rpb4
deletion destabilizes Rpb7 and facilitates Rpb7 dissociation.
Indeed, the Rpb4–Rpb7 interface is conserved (15) such that
stable chimeric heterodimers with Rpb4 and Rpb7 from various
species can be formed (14, 17, 26). Consistent with our model,
Rpb7 overexpression can suppress Rpb4 deletion defects (23,
27). In contrast to our findings, Rpb7 dissociation after Rpb4
deletion was explained earlier by core interaction primarily
through Rpb4 (5, 20, 28).
Rpb7 Forms a Conserved Wedge That Restrains the Clamp. The Rpb7
tip forms a wedge between the clamp, the linker, and the core
subunit Rpb6 (Fig. 2A). The tip partially fills a surface ‘‘pocket,’’
which corresponds to the end of the previously identified
potential RNA exit groove 1 (6). The pocket is lined by five
protein regions: three in Rpb1, and one each in Rpb2 and Rpb6
(Fig. 3). Rpb4?7 binding to the pocket thus holds together three
subunits and may stabilize the Pol II subunit assembly. The
pocket–Rpb7 interface is partially hydrophobic and conserved
among eukaryotes (Fig. 3), explaining why human Rpb4?7 can
functionally replace its yeast counterpart (29).
A corresponding pocket–tip interaction apparently exists in
eukaryotic Pol I and Pol III and in the archaeal RNA polymer-
ase. The tip domain is conserved in the Rpb7 homologs of Pol
I (10, 11), Pol III (9, 12), and archaeal polymerase (15). Similar
to Rpb7, the Rpb7 homolog of Pol I binds the common subunit
Rpb6 (11) and can dissociate from the core in a Pol I mutant
(30). The Rpb7 homolog of Pol III is also dissociable, and
disruption of its tip domain is lethal (9). Because core subunits
are either shared or homologous in all three eukaryotic enzymes
(31), the Rpb4?7 counterparts most likely bind similarly to the
cores. Thus the yeast 12-subunit Pol II is a good model for
Pol I and Pol III and for the enzymes in higher eukaryotes,
because Pol II subunits are highly conserved in sequence and
In the 12-subunit Pol II, the clamp adopts the closed confor-
mation observed in the core elongation complex structure (8),
consistent with electron microscopy in solution (20). Except for
the clamp closure, there are no gross changes in core structure
after Rpb4?7 binding. Modeling the clamp in the open states
observed in the free Pol II core structures (7) results in a clash
with the Rpb7 tip (Fig. 2A). Thus Rpb4?7 can only bind when
the clamp is closed, and the clamp can only open after Rpb4?7
has dissociated from the Pol II core. Residual space between the
closed clamp and Rpb4?7 only allows for slight changes in the
Implications for Transcription Initiation. Coupling between Rpb4?7
binding and clamp closure is relevant for transcription initiation
when promoter DNA is loaded onto polymerase. Given that the
DNA duplex would be loaded deeply into the cleft, between the
clamp and the wall as suggested (7), Rpb4?7 must dissociate
Table 1. X-ray diffraction data
Data set*NativeRpb4?7 SeMet
Unit cell axis,†Å
Resolution, Å (highest shell)
Rsym, % (highest shell)
No. of peaks in
220.4, 391.4, 282.2
223.0, 394.5, 284.4
0.9795 (Se peak)
93.8 (89.5)99.9 (100.0)
13 (5 Se, 8 Zn)
*Diffraction data were collected at the Swiss Light Source.
†Space group C2221.
counterpart structure (15) to the initial 4.2-Å difference Fourier map (green).
The map was phased with the Pol II core structure and is contoured at 2?. C?
features. The view corresponds to the ‘‘front’’ view of Pol II (6). (B) Selenome-
was calculated with anomalous data from a crystal with selenomethionine-
labeled Rpb4?7 (Table 1) and phases from the core model and is contoured at
with heights between 20.5? and 10.6? coincide with the location of methio-
nine side chains. Indicated as yellow spheres are sulfur atoms in methionine
were observed for Rpb4 methionines 1 and 114, which are in flexible regions.
The figure was prepared with O (53).
Structural analysis of the 12-subunit Pol II. (A) Fit of the Rpb4?7
Armache et al.
June 10, 2003 ?
vol. 100 ?
no. 12 ?
from the Pol II core for sufficient clamp opening. However,
Rpb4?7 could rebind after DNA melting and clamp closure.
Although Rpb4?7 can dissociate from the Pol II core in vitro (5),
it is unclear whether it dissociates in vivo. Our model reveals a
small core–Rpb7 interface, consistent with a transient interac-
tion, but the core–Rpb4?7 complex is more stable in other
species (17, 26, 28).
In an alternative scenario for initiation, Rpb4?7 persistently
bound to core would prevent promoter entry to the cleft, and
DNA could only bind far above the active center. After DNA
melting, the template strand, however, could pass the clamp, slip
into the cleft, and bind to the site formed by switch regions 1–3
as observed in the core elongation complex (8). Except for a
central part in switch 3, around Rpb2 residues 1120–1127,
switches 1–3 adopt a similar conformation in the 12-subunit Pol
II and are no longer flexible as in the free core structures (7).
Thus, interaction of Rpb4?7 with the Pol II core induces partial
formation of the binding site for the DNA template strand. The
flexible part of switch 3 may only get ordered after template-
strand binding or formation of an early DNA–RNA hybrid.
Induced folding of the central part of switch 3 by the growing
hybrid could underlie a transition that stabilizes the early
Promoter loading minimally requires assembly of TFIIB,
TFIIF, and the TATA box-binding protein (TBP) to DNA
regions upstream of the transcription start site (33, 34). Topo-
logical considerations predict that these factors interact with the
‘‘upstream face’’ of Pol II around the ‘‘dock’’ domain (Fig. 2B;
ref. 7). Rpb4?7 dramatically extends the upstream interaction
face (Fig. 2B), consistent with a role of Rpb4?7 in initiation
complex assembly. Indeed, Rpb4?7 stabilizes a minimal initia-
tion complex (35). Both Rpb4?7 and TFIIB bind to the Pol II
linker (Figs. 2 and 3; ref. 36), which may be a scaffold for
initiation complex assembly. Adjacent binding of Rpb4?7 and
TFIIB is consistent with an interaction between the archaeal
homologs of TFIIB and Rpb6 (37). In Pol III, the Rpb4 homolog
binds to a region corresponding to the linker (12), and it also
interacts with a TFIIB-related initiation factor (38). The Rpb7
is as described for Fig. 1. Cyan spheres and a pink sphere depict eight zinc ions and an active-site magnesium ion, respectively. A black line circles the clamp. The
linker to the CTD is indicated as a dashed line. In the lower-right corner, a schematic cut-away view is shown. A dashed line indicates the open clamp position
observed in form 2 of the Pol II core structure (7). (B) Pol II upstream interaction face. Shown in a view of the model from the ‘‘top’’ (6). The circle segment is
centered at the active site and has a radius that corresponds to the minimal distance between the TATA box and the transcription start site (85 Å, ?25 bp). The
saddle between the wall and the clamp and the assumed direction of RNA exit are indicated. A blue asterisk indicates a potential RNA-binding face of Rpb7 (15,
22). A key to subunit color is shown in the upper right corner. The figure was prepared with RIBBONS (54).
Architecture of the 12-subunit Pol II, coupling of Rpb4?7 binding and clamp closure, and upstream interaction face. (A) Ribbon model of Pol II. The view
alignments of protein regions are as described for A. Hs, Homo sapiens; Dm, Drosophila melanogaster; Sp, Schizosaccharomyces pombe; Sc, S. cerevisiae; Mj,
was prepared with RIBBONS (54).
Pocket–tip interaction. (A) Ribbon model of the Rpb7 tip binding with its two outermost loops (15) to the five protein regions (7) that line the pocket.
www.pnas.org?cgi?doi?10.1073?pnas.1030608100Armache et al.
homolog of Pol I also binds an initiation factor (39, 40). Thus
Rpb4?7 and its counterparts bridge the polymerase core with
initiation factors, and differences between them could contrib-
ute to promoter specificity. The roles of Rpb4?7 in initiation and
a possible structural role (see above) can account for transcrip-
tion shut off in an rpb4 deletion strain of yeast at high temper-
It is likely that promoter loading is topologically similar during
bacterial transcription initiation, which requires only the ? factor
and the core polymerase. The structural conservation of bacte-
rial and eukaryotic core RNA polymerases (44, 45) enables a
comparison of our model with recent structures of bacterial
RNA polymerase bound to ? (46, 47) and bound to ? and
upstream promoter DNA (48). In these structures, the ? factor
interacts with regions corresponding to the Pol II upstream face
where TFIIB, TFIIF, and TATA box-binding protein are pro-
posed to assemble. Although the ? factor shows sequence
similarity to Rpb4 (25), it is not a functional counterpart of
Rpb4?7, because Rpb4?7 and ? bind to different sites on the
polymerase surfaces. The structure of bacterial polymerase with
bound promoter shows a closed clamp and ordered switches, as
observed in our model of the initiation-competent Pol II.
Additionally, the bacterial complex shows promoter DNA out-
side the cleft, consistent with our alternative scenario for initi-
ation, in which duplex DNA is loaded and melted above the cleft.
eukaryotes but not in bacteria is reflected in its functions during
transitions within the transcription cycle. During initiation, the
Pol II CTD gets phosphorylated and remains phosphorylated
during RNA elongation. Dephosphorylation of the CTD, how-
ever, is required for Pol II recycling after termination, because
only unphosphorylated Pol II can rejoin an initiation complex.
CTD dephosphorylation is carried out by the phosphatase Fcp1
(49). Fcp1 is apparently recruited to the phosphorylated CTD by
Rpb4?7, because Rpb4 binds Fcp1 (50), and Rpb7 binds the
linker to the CTD, which is disordered in our crystals (Figs. 2 and
3). Fcp1 inhibits initiation complex assembly (51), maybe be-
cause Fcp1 and a general factor bind to the upstream face in a
mutually exclusive manner, ensuring complete CTD dephos-
phorylation before transcription reinitiation.
The 12-subunit Pol II model explains and suggests functional
roles of the Rpb4?7 complex and begins to extend our under-
standing of eukaryotic transcription toward the mechanism of
initiation. Our work further shows that the architecture of a
dissociable multiprotein complex can be determined by x-ray
analysis even at moderate resolution. The methods used and
developed here can be applied to complexes of Pol II with
proteins and nucleic acids to address mechanistic questions
raised by the complete Pol II model.
We thank C. Schulze-Briese and T. Tomizaki for help at the protein
crystallography beamline X06SA of the Swiss Light Source; M. Kimura
and A. Ishihama for the Rpb4?7 expression plasmid; C. Carles, M. Siaut,
and P. Thuriaux for sending manuscripts before publication; K.-P.
Hopfner for help with mass spectrometry; A. Meinhart, C. Buchen,
and other laboratory members for help; and Q. Eastman, D. Eick, R.
Grosschedl, K.-P. Hopfner, M. Meisterernst, R. Sachdev, and members
of the laboratory for critical reading of the manuscript. This work was
supported by Deutsche Forschungsgemeinschaft Research Grant
CR117-2?1, the European Molecular Biology Organization Young
Investigator Program, and the Fonds der Chemischen Industrie.
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