89
Transcription of the genetic information in all cells is carried
out by multisubunit RNA polymerases (RNAPs). Comparison
of the crystal structures of a bacterial and a eukaryotic RNAP
reveals a conserved core that comprises the active site and a
multifunctional clamp. Together with a further structure of
eukaryotic RNAP bound to DNA and RNA, these results
elucidate many aspects of the transcription mechanism,
including initiation, elongation, nucleotide addition, processivity
and proofreading.
Addresses
Institute of Biochemistry, Gene Center, University of Munich,
Feodor-Lynen-Strasse 25, 81377 Munich, Germany;
e-mail: cramer@lmb.uni-muenchen.de
Current Opinion in Structural Biology 2002, 12:89–97
0959-440X/02/$ — see front matter
© 2002 Elsevier Science Ltd. All rights reserved.
Abbreviations
bRNAP bacterial RNA polymerase
NTP nucleoside triphosphate
Pol I, II, III RNA polymerase I, II, III
RNAP RNA polymerase
yRNAP yeast RNA polymerase II
yRNAP-EC yeast RNA polymerase II elongation complex
Introduction
Multisubunit RNA polymerases (RNAPs) synthesize RNA
from a DNA template in the course of gene transcription.
Bacteria and archaea have one RNAP, whereas eukaryotes
have three RNAPs, responsible mainly for the synthesis of
ribosomal RNA (RNA polymerase I [Pol I]), pre-messenger
RNA (Pol II) and small RNAs including transfer RNAs
(Pol III). The complexity and large size of RNAPs (5–15
subunits, up to 0.6 MDa) posed formidable technical
challenges to structural biologists. Over the past few
years, however, X-ray crystallographic structures have
been determined of a bacterial RNAP from Thermus aquaticus
(bRNAP) at 3.3 Å resolution ([1]; reviewed in [2]), of
bRNAP bound to the inhibitor rifampicin at 3.3 Å resolution
[3
••
], of Pol II from the yeast Saccharomyces cerevisiae
(yRNAP) in two crystal forms at 2.8 Å and 3.1 Å resolution
[4
••
,5
••
], and of a yeast Pol II elongation complex
(yRNAP-EC) at 3.3 Å resolution [6
••
]. These structures
form a basis for understanding the function of all RNAPs
and for dissecting the transcription mechanism by site-
directed mutagenesis and further structural studies. In this
review, I describe the known RNAP structures and their
functional implications.
A technical tour de force
To solve the crystal structures of the large and asymmetric
bRNAP and yRNAP multiprotein complexes, several
obstacles had to be overcome ([1,4
••
,5
••
,7] and references
therein). Both enzymes had to be purified from large
quantities of cell culture without the benefit of over-
expression. In both cases, interpretation of the experimental
electron density maps relied on the placement of known
subunit structures and phase combination. In the case of
bRNAP, X-ray diffraction was weak, radiation sensitive
and anisotropic. The interpretation of maps with weak
sidechain electron density relied on noncrystallographic
symmetry averaging and data from selenomethionyl
bRNAP. In the case of yRNAP, a mutant yeast strain had to
be used to produce Pol II lacking two substoichiometric
subunits. Nonisomorphous and weakly diffracting initial
Pol II crystals were improved dramatically by a soaking
procedure that induced crystal shrinkage. Single heavy atoms
could not be detected in Patterson maps and initial phasing
therefore relied on heavy atom clusters. Single heavy
atom derivatives, needed for phasing to higher resolution,
could only be obtained with nonstandard compounds.
Model building was greatly facilitated by sequence markers,
including native zinc ions, mercury-labeled cysteines and,
most notably, selenomethionine, which could be partially
incorporated in yeast despite its toxicity [8].
General RNA polymerase architecture
The independently determined yRNAP and bRNAP
structures reveal that five ‘core’ subunits underlie a general
RNAP architecture. The two large subunits form the
central mass of the enzyme and opposite sides of a
positively charged cleft (Rpb1 and Rpb2 in yRNAP; β′ and
β in bRNAP; Table 1, Figure 1). The two large subunits
are anchored by two small core subunits that are involved
in RNAP assembly (Rpb3–Rpb11 heterodimer in yRNAP;
α homodimer in bRNAP; Figure 1). A fifth core subunit
(Rpb6 in yRNAP; ω in bRNAP) further buttresses and
stabilizes the large subunit [9–11] (Figure 1).
One side of the cleft (the Rpb1/β′ side) is formed by a
mobile ‘clamp’ (see below). The other side (the Rpb2/β
side) is formed by two domains: the ‘lobe’ and ‘protrusion’
domains in yRNAP, and the ‘β domains 2 and 3’ in bRNAP
(Figure 2). The active site is located at the floor of the
cleft, near the center of the enzyme (Figure 1). Beyond the
active site, the cleft is blocked by a ‘wall’ or ‘flap’ (Figure 2).
Just before the active site, a long α helix spans the cleft
(‘bridge’, ‘β′F helix’; Figure 1). This helix and the active
site line a perforation in the floor of the cleft (‘pore 1’,
‘secondary channel’; Figure 1), which widens towards the
exterior, creating an inverted funnel. The outer rim of the
funnel is lined by a pair of α helices in the largest subunit
(funnel region; Figure 2).
Conserved core
A total of 22 homology regions in the core subunits [11–14]
cluster around the active site. Most portions of these homology
regions are structurally conserved between bRNAP and
Multisubunit RNA polymerases
Patrick Cramer