RNA Polymerase II Structure

in Eukaryotes
Patrick Cramer
University of Munich, Munich, Germany

RNA polymerase II (pol II) carries out transcription of protein-
coding genes by catalyzing DNA-directed synthesis of messen-
ger RNA. Detailed three-dimensional structures are now
available for yeast pol II in free form, in form of a minimal
elongation complex with bound nucleic acids, in an inhibited
form with the bound toxin a-amanitin, and in complex with
two additional subunits, Rpb4 and Rpb7. Together with
functional data, these structures provide many insights into the
mechanism of mRNA transcription.
Architecture
A ten-polypeptide pol II core enzyme is sufficient to
elongate the RNA transcript. For transcription initiation,
an additional two-subunit Rpb4/7 complex, and several
general transcription factors are required. In the crystal
structures of the pol II core, the two large subunits, Rpb1
and Rpb2 (Table I), form the central mass of the enzyme,
and opposite sides of a positively charged “cleft” that
contains the active center (Figure 1). The two large
subunits are bridged on one side by a module of subunits
Rpb3, Rpb10, Rpb11, and Rpb12. Around the periphery
of the enzyme, Rpb5, Rpb6, and Rpb8 bind to Rpb1, and
Rpb9 binds to both Rpb1 and Rpb2. The structures lack
subunits Rpb4 and Rpb7, which form a stable hetero-
dimer that can dissociate from the yeast pol II core. The
structure of an archaeal Rpb4/7 counterpart revealed
that Rpb7 spans the elongated heterodimer and is organi-
zed in two domains. The Rpb4 homologue forms a con-
served hydrophobic interface with the Rpb7 homologue
at the connection between the two Rpb7 domains. Recent
structures revealed that the Rpb4/7 complex binds to the
pol II core via subunit Rpb7 and that it protrudes from
the pol II core surface near subunit Rpb6.
subunits Rpb9 and Rpb5, respectively. The end of the
cleft is blocked by a protein wall. The active center is at
the floor of the cleft, between the protrusion, the wall,
and the clamp. Before the active center and opposite of
the wall, a long bridge helix spans the cleft. The bridge
partially lines a pore in the active center, which widens
toward the other side of the enzyme, creating an inverted
funnel. The rim of the pore includes the highly conserved
aspartate loop of Rpb1 that binds a catalytic Mg2þ ion,
termed metal A. A second metal ion, metal B, is weakly
bound next to metal A further in the pore. Both metal
ions are accessible from one side.
The clamp is trapped in two different open states in the
free core structures, but is rotated and closed in the
structure of the pol II transcription –elongation complex
that comprises template DNA and the RNA transcript.
In this structure, the clamp binds the DNA template
strand with three out of five switch regions. The switch
regions form the base of the clamp, connecting to the
remainder of pol II. Upon clamp closure, the switches
change conformation or undergo folding transitions.
The closed conformation of the clamp is also observed
in the complete pol II that contains the Rpb4/7 complex.
The Rpb4/7 complex appears to form a wedge
between the clamp and subunit Rpb6, thus preventing
clamp opening.
The Rpb1 polypeptide chain protrudes below the
Rpb4/7 complex on the outside of pol II, and gets
disordered after a few residues. These residues of Rpb1
constitute the beginning of a flexible linker that connects
to the mobile C-terminal repeat domain (CTD). The
CTD, a unique feature of pol II, consists of repeats of a
heptapeptide with the consensus sequence Tyr-Ser-Pro-
Thr-Ser-Pro-Ser. A total of 26 and 52 CTD repeats are
found in yeast and human Rpb1, respectively.

Structural Elements
Mechanism
The Rpb1 side of the pol II cleft is formed by a mobile
clamp, whereas the Rpb2 side consists of the lobe and TRANSCRIPTION CYCLE
protrusion domains. The entrance to the cleft is formed The transcription cycle is divided into three phases:
between the upper and the lower jaw, which include initiation, elongation, and termination. Steps during

Encyclopedia of Biological Chemistry, Volume 3. q 2004, Elsevier Inc. All Rights Reserved. 770
 RNA POLYMERASE II STRUCTURE IN EUKARYOTES 771

TABLE I
Polypeptide Composition of Cellular RNA Polymerases
a
Pol I Pol II Pol III Archaea Bacteria Class

A190 Rpb1 C160 A0 þ A00 b0 Core

0 00
A135 Rpb2 C128 B (B þ B ) b Core
AC40 Rpb3 AC40 D a Core
AC19 Rpb11 AC19 L a Core
Rpb6 Rpb6 Rpb6 K v Core, common
Rpb5 Rpb5 Rpb5 H Common
Rpb8 Rpb8 Rpb8 Common
Rpb10 Rpb10 Rpb10 N Common
Rpb12 Rpb12 Rpb12 P Common
A14 Rpb4 C17 F Rpb4/7
A43 Rpb7 C25 E Rpb4/7
A12.2 Rpb9 C11 X Specific
A34.5 Specific
A49 Specific
C82 Specific
C34 Specific
C31 Specific

a
Core, sequence partially homologous in all RNA polymerases;
common, shared by all eukaryotic RNA polymerases; Rpb4/7, Rpb4/7
heterodimer and its structural counterparts.

initiation include promoter binding, DNA melting, and

synthesis of short RNA transcripts. The transition from
initiation to elongation is referred to as promoter escape,
and results in a stable elongation complex that is
characterized by an open DNA region, the transcription
bubble. The bubble contains the DNA –RNA hybrid, a
heteroduplex of eight or nine base pairs. The growing
RNA 30 -end is located at the end of the hybrid that is
engaged with the pol II active site. Pol II alone can
maintain an open transcription bubble, translocate
along the DNA template, synthesize RNA, and detect
errors in the nascent RNA. For all other steps, however,
pol II requires the help of additional proteins.
Several steps of the transcription cycle are
accompanied by phosphorylation or dephosphorylation
of the pol II CTD. During initiation, the CTD gets
phosphorylated, and the CTD phosphorylation pattern
changes during elongation. CTD phosphorylation pat-
terns govern specific interaction with RNA processing
factors, thereby coupling transcription to RNA proces-
sing. The CTD is flexibly linked to a region beyond the
saddle, from which RNA exits, consistent with its role in
coupling transcription to mRNA processing. Several
kinases and at least one phosphatase control the
phosphorylation state of the pol II CTD.
INITIATION
To bind and melt promoter DNA, pol II assembles
on DNA with the general transcription factors TFIIB,
FIGURE 1 RNA polymerase II structures. (A) Ribbon model of yeast
RNA polymerase II. The 12 subunits are shown in different colors. The
active site metal ion A is depicted as a pink sphere. Zinc ions are shown
as cyan spheres. (B) Schematic cut-away view of the pol II core
elongation complex. The view is related to the one in (A) by a 908
rotation around a vertical axis. The DNA template and nontemplate
strands are in blue and green, respectively, and the RNA is in red. Four
of the bases in the DNA template strand are indicated as sticks
protruding from the backbone. The yellow oval depicts the presumed
binding site for the incoming nucleoside triphosphate substrate.
-D, -E, -F, and -H. The general factors are involved in
sequence-specific promoter recognition (TFIIB, TFIID),
prevention of nonspecific DNA binding (TFIIF), and
DNA melting and CTD phosphorylation (TFIIE,
TFIIH). RNA synthesis initiates within the bubble.
The early transcribing complex is functionally
unstable. Short RNAs are frequently released and pol
II has to restart transcription (abortive cycling). There
is a decline in abortive transcription when the RNA
reaches a length of about four nucleotides, a transition
772 RNA POLYMERASE II STRUCTURE IN EUKARYOTES
termed “escape commitment.” RNA that has grown to
a length of at least four nucleotides is generally not
contacted by pol II anymore and is held in the
elongation complex by base pairing with the DNA
template strand. A second barrier in the transition
from initiation to elongation has to be overcome when
the RNA reaches a length of about ten nucleotides.
At this length, the 50 -end of the RNA is detached from
the DNA template strand, and is redirected to the pol
II “saddle” and an exit tunnel. A third transition of the
early elongation complex is reflected in the continued
requirement for ATP and TFIIH until the RNA is about
15 nucleotides long. This transition may reflect
successful positioning of all bubble-maintaining struc-
tural elements of pol II with respect to the bubble,
and detachment of RNA from the pol II surface. Two
possible RNA exit grooves have been suggested
beyond the saddle, and binding of RNA to the saddle
and to one of the exit grooves could account for an
additional gain in stability of the elongation complex.
Successful passage of early pol II elongation complexes
through all three transitions has been referred to as
“promoter clearance.”
ELONGATION
During elongation, downstream DNA enters pol II at
two mobile “jaws,” and extends through the cleft
toward the active site. Beyond the active site, a nine
base pair DNA – RNA hybrid extends upward, toward
the wall. The axes of the downstream DNA duplex and
the DNA – RNA hybrid heteroduplex enclose an angle of
, 908. The growing RNA 30 -end is located above the
pore, which allows entry of nucleoside triphosphate
substrates from below. In the crystal structure of the pol
II elongation complex, the incoming DNA duplex is
mobile and badly ordered. However, three nucleotides
before the active site, the DNA template strand becomes
well-ordered by binding to the bridge helix and to two
“switch” regions at the base of the clamp. A 908 twist
between subsequent nucleotides orients a DNA base
toward the active site for base pairing with an
incoming nucleotide.
The property of the polymerase to stay attached to
the template, even during transcription of long genes, is
often referred to as processivity. The major cause of
processivity is the high stability of the pol II elongation
complex, caused by tight binding of the DNA – RNA
hybrid. This stability can be accounted for by the highly
complementary hybrid-binding site on pol II, which is
partially created upon clamp closure and folding of the
switches. Several pol II structural elements are predicted
to maintain the hybrid and the transcription bubble
during elongation, including the fork loops, the top of
the wall, and three loops protruding from the edge of the
clamp, called rudder, lid, and zipper.
The nucleotide addition cycle during RNA chain
elongation begins with entry of the nucleoside tripho-
sphate (NTP) substrate, maybe together with metal B,
and its binding between the bridge helix and the end
of the hybrid, to form a base pair with the coding
DNA base. Correct orientation of the substrates and
metal ions would lead to synthesis of a new
phosphodiester bond and to the release of pyrophos-
phate, maybe together with metal B. The resulting
complex adopts the pre-translocation state, which
was apparently trapped in the pol II elongation
complex structure, with the RNA 30 -terminal nucleo-
tide occupying the NTP-binding site. Subsequent
translocation of nucleic acids would align the new
RNA 30 end with metal A, and would free the NTP-
binding site, preparing pol II for another cycle of
nucleotide addition.
Specificity for synthesizing RNA rather than DNA is
achieved by at least three mechanisms. First, the
discriminating 20 -OH group of the incoming NTP may
be hydrogen-bonded by a conserved pol II residue.
Second, 20 -OH groups of the last few nucleotides that
were incorporated into the growing RNA are directly
hydrogen-bonded by pol II residues. Finally, the active
center of pol II is complementary to the resulting DNA –
RNA hybrid duplex that adopts a conformation inter-
mediary between canonical A- and B-forms.
It is a mystery of the transcription mechanism how
rapid translocation of nucleic acids can be achieved
while nucleic acids are bound tightly by pol II. Hints for
understanding translocation are, however, provided by
the pol II structures. First, nucleic acids are only
contacted via their backbones; base interactions that
would impede translocation are not observed. Second,
long-range electrostatic attraction of the nucleic acid
backbones by positively charged protein groups in a
“second shell” may enable tight binding of nucleic acids
without restricting their movement. Finally, transloca-
tion may be accompanied by conformational changes in
pol II that could maintain some of the protein – nucleic
acid contacts during translocation. Structural compari-
sons suggest that one such conformational change may
be bending of the bridge helix.
Pol II elongation is inhibited by the cyclic octapeptide
a-amanitin, the toxin of the “death cap” mushroom.
a-Amanitin does not greatly influence NTP binding, and
a phosphodiester bond can still be formed when the
toxin is added to an elongation complex. However,
the rate of transcription is dramatically reduced. Only
several nucleotides are incorporated per minute. In the
structure of a pol II core – a-amanitin complex,
a-amanitin binds to the bridge helix, and could restrain
a possible movement of this helix, thereby blocking
conformational changes that are important for translo-
cation. However, the exact mechanism of pol II
inhibition by a-amanitin is not understood.

Pol II does not move along the DNA template in a
unidirectional manner, it rather oscillates between
movements forward and backward (“backtracking”).
During backtracking, RNA is extruded through the pol
II pore into the funnel. Backtracking can lead to
transcriptional pausing and arrest. Pausing is defined
as a temporary block to elongation, from which pol II
can escape by itself. In contrast, pol II cannot escape
from arrest without the help of the transcript cleavage
factor TFIIS, which stimulates a weak intrinsic nuclease
activity of pol II, which cleaves RNA. Access of TFIIS to
the pol II active site is apparently provided via the funnel
and pore. Backtracking and TFIIS action may also
underlie “proofreading” of the nascent transcript.
During proofreading, a misincorporated nucleotide
results in a mismatch base pair within the DNA – RNA
hybrid. The induced distortion of the hybrid destabilizes
the elongation complex and can lead to immediate
cleavage of a mononucleotide, or it can trigger in
backtracking, which results in threading of a short
stretch of RNA (comprising the misincorporated
nucleotide) into the pore and cleavage with the help
of TFIIS. Any proofreading reaction creates a new
RNA 30 -end at the active site, from which transcription
can resume.
TERMINATION
Transcription termination occurs in a reaction coupled
to RNA 30 -end processing. Most eukaryotic mRNA
precursors are cleaved in a site-specific manner in the
30 -untranslated region, followed by polyadenylation of
the upstream cleavage product. A large number of
proteins is involved in these reactions. The exact
mechanism of coupling between 30 -end processing and
transcription termination remains unclear. Termination
is accompanied by dephosphorylation of the pol II CTD,
but the precise timing of pol II dephosphorylation is also
unclear. Dephosphorylation is required for the reinitia-
tion of transcription, since pol II can only join an
initiation complex in its unphosphorylated form. The
CTD phosphatase Fcp1 plays a key role in pol II
dephosphorylation and recycling. Fcp1 binds to pol II
via Rpb4. Rpb4 apparently recruits Fcp1 to the vicinity
of the CTD since the Rpb4/7 complex binds near the
linker that connects the pol II core to the CTD.
Conservation
Pol II belongs to the family of cellular RNA polymerases,
which also includes the two other eukaryotic RNA
polymerases, pol I and pol III, and the bacterial and
archaeal RNA polymerases. All three eukaryotic RNA
polymerases share five common subunits (Table I). Four
RNA POLYMERASE II STRUCTURE IN EUKARYOTES 773
core subunits of pol II, Rpb1, Rpb2, Rpb3, and Rpb11,
as well as subunit Rpb9, all have close homologues in
pol I and pol III. Recent studies show that the Rpb4/7
complex of pol II also has structural and functional
counterparts in pol I and pol III, and in the archaeal
RNA polymerase. Since the 12 subunits of pol II are
either identical or homologous in all three eukaryotic
enzymes, the pol II structure is a good model for all
eukaryotic RNA polymerases. Comparison of pol II
with a bacterial RNA polymerase structure revealed that
five core subunits underlie a general RNA polymerase
architecture with an active center cleft. Twenty-two
regions of sequence homology cluster around the active
site and generally adopt the same structure in all cellular
RNA polymerases. The structurally conserved core
includes the functional elements of the active center,
indicating that all cellular RNA polymerases share
common mechanistic features. Homologues for all pol
II subunits (except Rpb8) are found in archaeal RNA
polymerases. Thus the overall structure of archaeal
RNA polymerases is very similar to that of pol II except
several external domains.
The pol II structure is strikingly different from
structures of the many single subunit DNA and RNA
polymerases. However, in functional complexes of these
diverse enzymes, nucleic acids take a similar course
through the active center. The entering DNA duplex
encloses an angle of , 908 with the exiting template–
product duplex. At the location of the bend, subsequent
DNA template bases are twisted. This twist aligns the
coding base with the binding site for the incoming
nucleoside triphosphate substrate. The nucleoside tri-
phosphate enters through an opening that is found in all
polymerases, and generally binds between an a-helix
and two catalytic metal ions. The exiting template–
product duplex is bound from the minor groove side in
all polymerases. Conformational changes upon nucleic
acid binding have been detected for several different
polymerases, but the nature of this “induced fit” differs.
SEE ALSO THE FOLLOWING ARTICLES
Recombination: Heteroduplex and Mismatch Repair
in vitro † RNA Polymerase I and RNA Polymerase III in
Eukaryotes † RNA Polymerase II and Basal Transcrip-
tion Factors in Eukaryotes † RNA Polymerase II
Elongation Control in Eukaryotes
GLOSSARY
backtracking Reverse movement of pol II along DNA and RNA.
DNA–RNA hybrid 8 –9 base pair double-stranded heteroduplex
formed between the DNA template strand and the RNA transcript
within the transcription bubble.
downstream DNA DNA that is going to be transcribed.
Rpb1–Rpb12 RNA polymerase B (II) polypeptide subunits 1– 12.
774 RNA POLYMERASE II STRUCTURE IN EUKARYOTES
transcription bubble Open DNA region in the active center of pol II.
translocation Movement of pol II by a step of one base pair along the
DNA template.
upstream DNA DNA that has been transcribed.
FURTHER READING
Armache, K.-J., Kettenberger, H., and Cramer, P. (2003). Architecture
of the initiation-competent 12-subunit RNA polymerase II. Proc.
Natl. Acad. Sci. USA 100, 6964–6968.
Bushnell, D. A., and Kornberg, R. (2003). Complete 12-subunit
RNA polymerase II at 4.1Å resolution: Implications for the
initiation of transcription. Proc. Natl. Acad. Sci. USA 100,
6969–6973.
Cramer, P. (2002). Multisubunit RNA polymerases. Curr. Opin. Struct.
Biol. 12, 89–97.
Cramer, P., Bushnell, D. A., Fu, J., Gnatt, A. L., Maies-Davis, B.,
Thompson, N. E., Burgess, R. R., Edwards, A. M., David, P. R., and
Komberg, R. D. (2000). Architecture of RNA polymerase II and
implications for the transcription mechanism. Science 288,
640–649.
Cramer, P., Bushnell, D. A., and Kornberg, R. D. (2001). Structural
basis of transcription: RNA polymerase II at 2.8 angstrom
resolution. Science 292, 1863–1876.
Gnatt, A. L., Cramer, P., Fu, J., Bushnell, D. A., and Kornberg, R. D.
(2001). Structural basis of transcription: An RNA polymerase II
elongation complex at 3.3Å resolution. Science 292, 1876–1882.
Woychik, N. A., and Hampsey, M. (2002). The RNA polymerase II
machinery: Structure illuminates function. Cell 108, 453–463.
BIOGRAPHY
Patrick Cramer is Assistant Professor of Biochemistry at the Institute of
Biochemistry and Gene Center of the University of Munich. His
research interest is in the structural biology of the eukaryotic mRNA
transcription machinery. He obtained a Ph.D. from Heidelberg
University and the European Molecular Biology Laboratory, and
carried out postdoctoral research at Stanford University. For his work
on the structure determination of RNA polymerase II, he was elected
EMBO Young Investigator in 2000 and received the GlaxoSmithKline
Science Award in 2002.