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Structure of Eukaryotic RNA Polymerases P. Cramer, K.-J. Armache, S. Baumli, S. Benkert, F. Brueckner, C. Buchen, G.E. Damsma, S. Dengl, S.R. Geiger, A.J. Jasiak, A. Jawhari, S. Jennebach, T. Kamenski, H. Kettenberger, C.-D. Kuhn, E. Lehmann, K. Leike, J.F. Sydow, and A. Vannini Gene Center Munich and Center for Integrated Protein Science CIPSM, Department ¨ of Chemistry and Biochemistry, Ludwig-Maximilians-Universit¨at Munchen, 81377 Munich, Germany; email:
[email protected]
Annu. Rev. Biophys. 2008. 37:337–52
Key Words
First published online as a Review in Advance on February 7, 2008
gene transcription, RNA synthesis, nucleic acid polymerase, X-ray crystallography, structural biology, multiprotein complex
The Annual Review of Biophysics is online at biophys.annualreviews.org This article’s doi: 10.1146/annurev.biophys.37.032807.130008 c 2008 by Annual Reviews. Copyright All rights reserved 1936-122X/08/0609-0337$20.00
Abstract The eukaryotic RNA polymerases Pol I, Pol II, and Pol III are the central multiprotein machines that synthesize ribosomal, messenger, and transfer RNA, respectively. Here we provide a catalog of available structural information for these three enzymes. Most structural data have been accumulated for Pol II and its functional complexes. These studies have provided insights into many aspects of the transcription mechanism, including initiation at promoter DNA, elongation of the mRNA chain, tunability of the polymerase active site, which supports RNA synthesis and cleavage, and the response of Pol II to DNA lesions. Detailed structural studies of Pol I and Pol III were reported recently and showed that the active center region and core enzymes are similar to Pol II and that strong structural differences on the surfaces account for gene class-specific functions.
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POL II STRUCTURE
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Contents INTRODUCTION . . . . . . . . . . . . . . . . . POL II STRUCTURE . . . . . . . . . . . . . . THE ELONGATION COMPLEX AND NUCLEOTIDE INCORPORATION . . . . . . . . . . . . . OVERCOMING OBSTACLES DURING ELONGATION . . . . . . THE POL II INITIATION COMPLEX . . . . . . . . . . . . . . . . . . . . . . STRUCTURAL STUDIES OF POL I . . . . . . . . . . . . . . . . . . . . . . . STRUCTURAL STUDIES OF POL III . . . . . . . . . . . . . . . . . . . . . . CONCLUSIONS AND OUTLOOK . . . . . . . . . . . . . . . . . . . . .
338 338
341 344 345 345 346 346
INTRODUCTION
Gene transcription: the process of RNA synthesis based on a DNA template DNA-dependent RNA polymerase: an enzyme that uses a DNA template to synthesize a complementary copy of RNA Pol I, Pol II, and Pol III: RNA polymerase I, II, III Rpb: RNA polymerase B (II) subunit
338
Gene transcription in eukaryotic cells is carried out by the three different DNAdependent RNA polymerases Pol I, Pol II, and Pol III. Pol I produces ribosomal RNA, Pol II synthesizes messenger RNAs and small nuclear RNAs, and Pol III produces transfer RNAs and other small RNAs. A fourth RNA polymerase, Pol IV, which was recently discovered in plants, is not included here, as its composition and structure are currently unknown. The RNA polymerases are multisubunit enzymes. Pol I, II, and III comprise 14, 12, and 17 subunits, respectively, and have a total molecular weight of 589, 514, and 693 kDa, respectively. Ten subunits form a structurally conserved core, and additional subunits are located on the periphery. Comprehensive reviews that summarized structural studies of Pol II were published four years ago (21, 35). Here we provide an up-todate catalog of structural studies of multisubunit RNA polymerases to facilitate access to the structural information. We emphasize recent structures and, in particular, recent structural studies of Pol I and Pol III, which allow for a first structural comparison of the three RNA polymerases. Cramer et al.
Pol II consists of a 10-subunit core enzyme and a peripheral heterodimer of subunits Rpb4 and Rpb7 (Rpb4/7 subcomplex, Table 1). The core enzyme comprises subunits Rpb1, Rpb2, Rpb3, and Rpb11, which contain regions of sequence and structural similarity in Pol I, Pol III, bacterial RNA polymerases (72, 82), and the archaeal RNA polymerase (49). The Pol II core also comprises subunits Rpb5, Rpb6, Rpb8, Rpb10, and Rpb12, which are shared between Pol I, II, and III (common subunits, Table 1). Counterparts of these common subunits except Rpb8 exist in the archaeal polymerase, but only a counterpart of Rpb6 exists in the bacterial enzyme (60). Finally, homologues of the core subunit Rpb9 exist in Pol I and Pol III, but not in the archaeal or bacterial enzyme. Initial electron microscopic studies of Pol II revealed the overall shape of the enzyme (26) (Table 2). The core Pol II could subsequently be crystallized, leading to an electron density map at 6 A˚ resolution (32). Crystal improvement by controlled shrinkage and phasing at 3 A˚ resolution resulted in a backbone model of the Pol II core (23). This revealed that Rpb1 and Rpb2 form opposite sides of a positively charged active center cleft, whereas the smaller subunits are arrayed around the periphery. Refined atomic structures of the core Pol II were obtained in two different conformations and revealed domain-like regions within the subunits, as well as surface elements predicted to have functional roles (24) (Figure 1 and Figure 2). The active site and the bridge helix, which spans the cleft, line a pore in the floor of the cleft. The Rpb1 side of the cleft forms a mobile clamp, which was trapped in two different open states in the free core structures (24) but was closed in the structure of a core complex that included DNA and RNA (34). The mobile clamp is connected to the body of the polymerase by five switch regions that show conformational variability. The Rpb2 side of the cleft consists
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Table 1
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RNA polymerase subunits
RNA polymerase
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Ten-subunit core
Rpb4/7 subcomplex TFIIF-like subcomplexa Pol III-specific subcomplex
Number of subunits
Pol I
Pol II
Pol III
A190
Rpb1
C160
A135
Rpb2
C128
AC40
Rpb3
AC40
AC19
Rpb11
AC19
A12.2
Rpb9
C11
Rpb5 (ABC27)
Rpb5
Rpb5
Rpb6 (ABC23)
Rpb6
Rpb6
Rpb8 (ABC14.5)
Rpb8
Rpb8
Rpb10 (ABC10α)
Rpb10
Rpb10
Rpb12 (ABC10β)
Rpb12
Rpb12
A14
Rpb4
C17
A43
Rpb7
C25
A49
(Tfg1/Rap74)
C37
A34.5
(Tfg2/Rap30)
C53
–
–
C82
–
–
C34
–
–
C31
14
12
17
a
The two subunits in Pol I and Pol III are predicted to form heterodimers that resemble part of the Pol II initiation/ elongation factor TFIIF, which is composed of subunits Tfg1, Tfg2, and Tfg3 in Saccharomyces cerevisiae, and of subunits Rap74 and Rap30 in human.
of the lobe and protrusion domains. Rpb2 also forms a protein wall that blocks the end of the cleft. The Pol II core structures lacked subunits Rpb4 and Rpb7, which can dissociate from the yeast enzyme (28). A structure of the archaeal homologue of the Rpb4/7 heterodimer showed that Rpb7 contains an N-terminal domain, later called the tip domain, and a C-terminal domain that includes an oligosaccharide-binding (OB) fold (70). The approximate location of Rpb4/7 on the core polymerase was first determined by electron microscopy (EM) of two-dimensional crystals (40). Later, EM analysis of single particles revealed a closed clamp and showed that the Rpb4/7 subcomplex protrudes from outside the core enzyme below the clamp (20). A different open-closed transition that involved the polymerase jaws was observed by EM of two-dimensional crystals (5). Crystal-
lographic backbone models of the complete Pol II then revealed the exact position and orientation of Rpb4/7 and showed that it formed a wedge between the clamp and the linker to the unique tail-like C-terminal repeat domain (CTD) of the polymerase (2, 11). The crystal structure of free Rpb4/7 (Figure 1d ), together with an improved resolution of the complete Pol II crystals, finally enabled refinement of a complete atomic model of Pol II (3) (Figure 1b). The CTD of Pol II is flexibly linked to the core enzyme and consists of heptapeptide repeats of the consensus sequence YSPTSPS. The CTD integrates nuclear events by binding proteins involved in mRNA biogenesis (for reviews see References 9, 37, and 56). CTD-binding proteins recognize a specific CTD phosphorylation pattern, which changes during the transcription cycle. Structural and functional studies of CTD-binding
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EM: electron microscopy
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Structural information on eukaryotic RNA polymerases Methoda
Resolutionc ˚ (A)
PDB code(s)
Reference(s)
Pol II subunits Rpb9 N-terminal domain
NMR
–
1qyp
(76)
Rpb10
NMR
–
1ef4
(54)
Rpb5
X-ray
1.9
1dzf
(71)
Rpb4/7 yeast
X-ray
2.3
1y14
(3)
Rpb4/7 human
X-ray
2.7
2c35
(58)
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Core Pol IIb Free
EM (2D)
16.0
–
(26)
+TFIIB or TFIIE
EM (2D)
15.7
–
(52)
Core ECb
EM (2D)
20
–
(62)
Free
X-ray
6.0
–
(32)
Free (form 1)
X-ray
3.1
1i3q
(23, 24)
Free (form 2)
X-ray
2.8
1i50
(24)
EC (tailed-template)
X-ray
3.3
1i6h
(34)
+α-amanitin
X-ray
2.8
1k83
(10)
Core EC
X-ray
3.6
1sfo
(81)
Core EC +NTP (insertion site) Core EC +NTP (entry site)
X-ray
4.25 3.5
1r9s 1r9t
(80)
Free +ATP +GTP +UTP +CTP +2 dATP
X-ray
3.2 3.0 2.3 3.3 3.4
1twa 1twc 1twf 1twg 1twh
(80)
Free core, 150 mM Mn2+ Core EC +GTP, 5 mM Mg2+ +2 dGTP, 5 mM Mg2+ +GMPCPP, 5 mM Mg2+ +2 dUTP, 150 mM Mg2+ +GMPCPP, 150 mM Mg2+ +2 dUTP, 5 mM Mg2+ +UTP (updated) +UTP, 150 mM Mg2+
X-ray
3.4 3.95 3.4 3.5 3.0 3.36 3.6 4.3 3.4
2nvy 2e2h 2e2i 2e2j 2nvq 2nvt 2nvx 2nvz 2yu9
(77)
Core +TFIIB
X-ray
4.5
1r5u
(12)
–
(40)
–
(20)
Complete Pol IIb Free
EM (2D)
Free
EM (cryo)
24 18
Free (backbone)
X-ray
4.2
1nt9
(2)
Free (backbone)
X-ray
4.1
1nik
(11)
Free (atomic)
X-ray
3.8
1wcm
(3)
+TFIIF
EM (cryo)
–
(19)
+TFIIS
X-ray
3.8
1pqv
(41)
EC
X-ray
4.0
1y1w
(42)
EC +TFIIS
X-ray
3.8
1y1v
(42)
18
(Continued ) 340
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Table 2
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(Continued )
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Methoda
Resolutionc ˚ (A)
PDB code(s)
Reference(s)
EC +NTP (inactive preinsertion-like state)
X-ray
4.5
1y77
(42)
CPD-damaged EC complex A complex B complex C complex D
X-ray
3.8 4.0 3.8 3.8
2ja5 2ja6 2ja7 2ja8
(8)
Cisplatin-damaged EC
X-ray
3.8
2r7z
(25)
RNA template-product complex
X-ray
3.8
2r92
(51)
HDV RNA hairpin
X-ray
4.0
2r93
(51)
Free human Pol II
EM
EMD-1283/1284
(46)
20
Pol II CTD peptidesb Free heptapeptide repeat
NMR
–
–
(36)
Peptide with eight repeats
NMR
–
–
(13)
Cyclic model peptides
NMR
–
–
(48)
Complex with WW domain
X-ray
1.84
1f8a
(75)
Complex with capping enzyme
X-ray
2.7
1p16
(29)
Complex with Pcf11 CIDb
X-ray
2.2 2.1
1sza 1sz9
(55)
Complex with Scp1
X-ray
2.05 1.8
2ghq 2ght
(83)
Pol I Free
EM (2D)
40
–
(65)
Free
EM (cryo)
34
–
(7)
Free
EM (cryo negative stain)
22
–
(27)
Free
EM (cryo)
11.9
EMD-1435
(47)
A49/34.5
EM (cryo)
25
–
(47)
Core
Homology model
–
–
(47)
A14/43
X-ray
2rf4
(47)
EMD-1322
(30)
–
(39)
2ckz
(39)
3.10
Pol III Free
EM (cryo)
Core
Homology model
C17/25
X-ray
17 – 3.2
a X-ray, X-ray crystallography; NMR, nuclear magnetic resonance; EM, electron microscopy (2D, two-dimensional electron crystallography; stain, negative staining; cryo, single particle cryo-EM). b Core, 10-subunit Pol II core enzyme; complete, complete 12-subunit Pol II; EC, elongation complex; CTD, C-terminal repeat domain of Pol II; CID, C-terminal repeat domain interacting domain. c Resolutions are not always comparable since different data cut-off criteria were used in different studies both in electron microscopy and X-ray crystallography.
and CTD-modifying proteins and their complexes with CTD peptides elucidated CTD structure and revealed some of the mechanisms underlying CTD function (Table 2).
THE ELONGATION COMPLEX AND NUCLEOTIDE INCORPORATION During the elongation phase of transcription, the polymerases move along a DNA template
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a
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b
RNA polymerase I
c
RNA polymerase II
RNA polymerase III
EM density
EM density
C82/34/31 C17/25
Rpb4/7
A14/43
C53/37
A49/34.5
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core Pol I model
d
core Pol III model
core Pol II
C OB domain
OB domain
OB domain
N
Tip domain
C
H*
H1
HRDC domain Tip-associated domain
Rpb4/7 (Pol II)
A14/43 (Pol I)
N
C
N
Tip-associated domain
C
N
C
N
Tip domain
Tip domain
HRDC domain Tip-associated domain
C17/25 (Pol III)
Figure 1 RNA polymerase structures. (a) Pol I hybrid structure (47). (b) Ribbon model of the refined complete Pol II crystal structure (3). (c) Pol III EM structure (30) with the Pol II-based homology model and the crystal structure of C17/25 (39) fitted as published in Reference 30. (d ) Rpb4/7 subcomplex structures: A14/43 (left), Rpb4/7 (center) (3), C17/25 (right).
and synthesize a complementary chain of ribonucleotides. RNA extension begins with binding of a nucleoside triphosphate (NTP) substrate to the transcription elongation complex (EC) that is formed by the polymerase, DNA, and RNA. Catalytic addition of the nucleotide to the growing RNA 3 end then releases a pyrophosphate ion. Finally, translocation of DNA and RNA frees the substrate site for binding of the next NTP. The EC is characterized by an unwound DNA region, the transcription bubble. The bubble contains a short hybrid duplex formed between the DNA template strand and the RNA product emerging from the active site. The mechanism of RNA elongation was elucidated by structural studies of Pol II– 342
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nucleic acid complexes (Figure 2). EM first revealed the point of DNA entry to the Pol II cleft (62). The first crystal structure of a Pol II–nucleic acid complex was that of the core Pol II transcribing a tailed template DNA (34), which allows for promoter-independent transcription initiation. This structure revealed downstream DNA entering the cleft and a 8 to 9 base pair DNA-RNA hybrid in the active center. Comparison with the high-resolution core Pol II structure (24) revealed protein surface elements predicted to play functional roles. Later, polymerase EC structures utilized synthetic DNA-RNA scaffolds (42, 81) and revealed the exact location of the downstream DNA and several nucleotides upstream of the hybrid. Mechanisms were
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a
b
A
Transcription
B
Bridge helix
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Active site metal A
DNA template DNA non-template RNA
NTP (insertion site) NTP (inactive pre-insertion state) Mismatched NTP (entry site)
c Wall
Zipper
180°
Rudder
Switch 3
Fork loop 1
Switch 2 Switch 1
Fork loop 2
βDloopII
Bridge helix Trigger loop
Functional Pol II elements
Figure 2 Structure of the Pol II elongation complex (EC). (a) Overview of the EC structure (42, 77, 80). (b) Superposition of NTP-binding sites [red, insertion site; violet, entry site (42, 77, 80); pink, inactive preinsertion-like state (42, 77, 80)]. (c) Functional Pol II surface elements in the EC.
suggested for how Pol II unwinds downstream DNA and how it separates the RNA product from the DNA template at the end of the hybrid. Although Pol II generally uses DNA as a template, there is also evidence that Pol II can use RNA templates. Recent structures showed that an RNA template-product duplex can bind to the site normally occupied by the DNA-RNA hybrid and provided the structural basis for the phenomenon of RNAdependent RNA synthesis by Pol II (51). Additional structures of Pol II ECs included the NTP substrate (42, 77, 80). These studies suggested how the polymerase selects the correct NTP and how it incorpo-
rates a nucleotide into RNA. The NTP was crystallographically trapped in the insertion site (77, 80), which is apparently occupied during catalysis, but also in an overlapping, slightly different location, suggesting an inactive NTP-bound preinsertion state of the enzyme (42). Both NTPs form Watson-Crick interactions with a base in the DNA template strand. Binding of the NTP to the insertion site involves folding of the trigger loop (77), a mobile part of the active center first observed in free bacterial RNA polymerase (72), and in the Pol II-TFIIS complex (41). Folding of the trigger loop closes the active site and may be involved in
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selection of the correct NTP. The NTP complex structures revealed contacts of the nucleotide with the polymerase, which explain discrimination of ribonucleotides against deoxyribonucleotides, and provided insights into the selection of the nucleotide complementary to the templating DNA base. Catalytic nucleotide incorporation apparently follows a two-metal ion mechanism suggested for all polymerases (69). The Pol II active site contains a persistently bound metal ion (metal A) and a second, mobile metal ion (metal B) (24). Metal A is held by three invariant aspartate side chains and binds the RNA 3 end (24), whereas metal B binds the NTP triphosphate moiety (80). Recent studies of functional complexes of the bacterial RNA polymerase revealed the close conservation of the EC structure (73) and provided additional insights into nucleotide incorporation (74). As for Pol II, NTP binding to the insertion site can induce folding of the trigger loop. In the presence of the antibiotic streptolydigin, however, the NTP binds in the inactive, preinsertion state, in which the triphosphate and metal B are too far from metal A to permit catalysis. This finding supported a twostep mechanism of nucleotide incorporation (42, 74). The NTP would first bind in the inactive state to an open active center conformation. Complete folding of the trigger loop then leads to closure of the active center, delivery of the NTP to the insertion site, and catalysis. An alternative model for nucleotide addition involves binding of the NTP to a putative entry site in the pore, in which the nucleotide base is oriented away from the DNA template, and rotation of the NTP around metal ion B directly into the insertion site (80).
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OVERCOMING OBSTACLES DURING ELONGATION During active transcription, Pol II must overcome intrinsic DNA arrest sites, which are generally rich in A-T base pairs and pose a natural obstacle to transcription. At such 344
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sites, Pol II moves backward along DNA and RNA, resulting in extrusion of the RNA 3 end through the polymerase pore beneath the active site and transcriptional arrest. The RNA cleavage stimulatory factor TFIIS can rescue an arrested polymerase by creating a new RNA 3 end at the active site from which transcription can resume. The mechanism of TFIIS function was elucidated with the structures of Pol II and a Pol II EC in complex with TFIIS (41, 42). TFIIS inserts a hairpin into the polymerase pore and complements the active site with acidic residues, changes the enzyme conformation, and repositions the RNA transcript (41, 42). These studies supported the idea that the Pol II active site is tunable, as it can catalyze different reactions, including RNA synthesis and RNA cleavage (41, 68). Other obstacles to transcription are bulky lesions in the DNA template strand. Recent structural studies of Pol II ECs that contain lesions in the template strand unraveled the mechanisms of polymerase stalling at two different bulky lesions, a UV light-induced thymine-thymine cyclobutane pyrimidine dimer (CPD) (8) and a guanineguanine intrastrand cross-link induced by the anticancer drug cisplatin (25). Cells efficiently eliminate CPDs and cisplatin cross-links by transcription-coupled DNA repair (TCR), which begins with Pol II stalling at the lesion. TCR continues with assembly of the nucleotide excision repair machinery, removal of a lesion-containing DNA fragment, and repair of the resulting DNA gap. The structural studies revealed that the structure of the EC is generally not influenced by the presence of the lesions, except for some changes in the downstream DNA, arguing against allosteric models for the assembly of the repair machinery during TCR. The studies also demonstrated a translocation barrier for bulky dinucleotide lesions that impairs their delivery to the active site. The important conclusion from these studies was that the detailed mechanisms of transcriptional stalling at two different
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dinucleotide lesions differ. The cisplatin lesion cannot overcome the translocation barrier, leading to polymerase stalling. However, the CPD can overcome the barrier and stably binds in the active site. Inefficient AMP incorporation occurs at both lesions by an apparently template-independent mechanism, but only for the CPD does this enable binding of the lesion at the active site via formation of a stable thymine-adenine base pair. Opposite the CPD, UMP misincorporation occurs, which finally stalls Pol II because the resulting mismatch impairs further translocation of nucleic acids. These studies also showed that Pol II can, under certain artificial conditions, bypass a distorting dinucleotide lesion, and suggested that it is currently impossible to predict the mechanisms of transcriptional stalling or mutagenesis at other types of lesions.
THE POL II INITIATION COMPLEX For transcription initiation, Pol II assembles with the initiation factors TFIIB, -D, -E, -F, and -H into an initiation complex at promoter DNA. Our current understanding of the architecture of the initiation complex was reviewed recently (4, 22, 35). Briefly, current models of initiation assume that the promoter DNA is first bound outside the Pol II cleft. Upon DNA melting, the template single strand is predicted to slip inside the Pol II cleft and bind near the active site for the determination of the transcription start site and the initiation of RNA synthesis. The mechanisms underlying these events, however, are poorly understood. The location of initiation factors along promoter DNA was revealed by site-specific DNA-protein cross-linking (6, 15, 44, 59). With the use of photo-cross-linking and radical probing, the N-terminal domain of TFIIB was located to the polymerase dock domain (16), and the C-terminal domain of TFIIB was placed over the polymerase cleft and wall (17). Crystallographic analysis of the Pol II
core-TFIIB complex confirmed that the Nterminal TFIIB zinc ribbon domain binds the dock domain and showed that a region between the N- and C-terminal domains of TFIIB, the B-finger, extended into the active center (12). EM of the Pol II-TFIIF complex suggested that the second largest TFIIF subunit extends along the cleft, whereas density at the Rpb4/7 subcomplex was attributed to the TFIIF largest subunit (19). A recent crosslinking study revealed an interaction of TFIIF with the opposite side of the cleft (18). It is likely that TFIIF, similar to TFIIB, extends with a loop into the active center (17, 31). Early EM placed the initiation factor TFIIE at the downstream jaws of Pol II (52), whereas cross-linking studies recently found an interaction of TFIIE with the clamp domain (18). TFIIH is located near downstream DNA (44, 59). Finally, studies of a complete Pol II complex with an RNA aptamer showed how transcription initiation can be inhibited by a small RNA (43). The RNA inhibitor binds the active center cleft and apparently prevents loading of the DNA into the cleft during initiation (43).
STRUCTURAL STUDIES OF POL I Compared with Pol II, little structural information is available for Pol I and Pol III. The fold of the 10-subunit core enzymes is conserved to a large extent, and the active center regions are highly similar in all three enzymes. Pol I and Pol III also contain counterparts of the Rpb4/7 subcomplex, A14/43 and C17/25, respectively, but there is hardly any sequence conservation between the corresponding subunits in these heterodimers (38, 57, 61, 63, 66, 67). Pol I further contains the specific subunits A49 and A34.5, and Pol III additionally contains the specific subunits C82, C53, C37, C34, and C31. For Pol I, the overall shape and dimensions were first revealed by EM analysis of two-dimensional crystals (65). Subsequent cryo-EM at 34 A˚ resolution [Fourier shell
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correlation (FSC) = 0.5] visualized a stalk containing A14/43 and densities for A49 and A34.5 over the central cleft (7, 61). Later EM analysis with cryo-negative staining at 22 A˚ (FSC = 0.5) confirmed the stalk but not the location of A49 and A34.5 (27). Recently, a 12 A˚ (FSC = 0.5) cryo-EM structure was reported for yeast Pol I, as well as a homology model for the core enzyme, and the crystal structure of the subcomplex A14/43 (47). In the resulting hybrid structure of Pol I (Figure 1a), A14/43, the clamp, and the dock domain contribute to a unique surface interacting with promoter-specific initiation factors, in particular Rrn3. A14/43 lacks the HRDC domain present in Rpb4/7 and C17/25 (Figure 1d ). Subunits A49 and A34.5 form a heterodimer near the enzyme funnel that is important for normal elongation activity and is predicted to be partly related to the Pol II initiation/elongation factor TFIIF.
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STRUCTURAL STUDIES OF POL III An interaction map between Pol III subunits was derived by yeast two-hybrid analysis and copurification assays (reviewed in References 14, 33, 64). The five Pol III–specific subunits apparently form two subcomplexes. Subunits C82, C34, and C31 form a trimeric subcomplex involved in initiation (78, 79), whereas C37 and C53 form another subcomplex involved in termination and reinitiation (30, 50, 53). The first structural information for Pol III has recently become available and includes a homology model for the 10-subunit core enzyme (39), the crystal structure of the C17/25 subcomplex (39), and a 17 A˚ EM structure (30) (Figure 1c,d ). Compared with Pol I and Pol II, Pol III shows a structurally different upstream face for specific initiation complex assembly during promoter selection. The Pol III upstream face includes the C82/34/31 subcomplex (30), which is involved in initiation,
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and a HRDC domain in subunit C17 that adopts a position different from its Pol II counterpart, Rpb4/7 (39). The C37/53 heterodimer may adopt a position on the outside of the core enzyme near the lobe and funnel (30) and may be the counterpart of the A49/34.5 heterodimer in Pol I (47). Mass spectrometry is consistent with a subcomplex architecture of Pol III that includes a 10subunit Pol II-like core; the peripheral heterodimers C17/25, C37/53, and C82/34; and subunit C31, which bridges between C82/34, C17/25, and the core (53).
CONCLUSIONS AND OUTLOOK The complexity and large size of multisubunit RNA polymerases have prevented elucidation of their structure for a long time. The first detailed insights into the architecture and possible mechanism of multisubunit RNA polymerases were obtained around the millennium, when the core Pol II crystal structures (23, 24, 34) and structural studies of the related bacterial enzyme (45, 82) became available. In the following years, the discovery of many additional structures of Pol II complexes, and structures of the bacterial enzyme that are not reviewed here, elucidated different aspects of transcription. Only over the past two years, more detailed structural information for Pol I and Pol III became available and revealed similarities and differences between the three eukaryotic RNA polymerases that emerged to match different functional properties and requirements. To explain the detailed differences in structure and function of these enzymes, it is necessary to also investigate Pol I and Pol III crystallographically, which is a tremendous experimental challenge. In addition, many aspects of the Pol II transcription mechanism remain unresolved, and more complex structures are required to clarify them. Among these open issues are the mechanisms of initial promoter binding and melting, initial transcription, translocation of nucleic acids after nucleotide addition, and
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termination, to name a few. A long-term goal of structural biology on RNA polymerases remains to obtain clues for how these en-
zymes are regulated by coregulatory assemblies, which transfer signals from transcriptional activators and repressors.
SUMMARY POINTS 1. Crystallographic studies of Pol II have elucidated the structure and conformational flexibility of a central biological multiprotein machine.
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2. Structural studies of Pol II complexes with nucleic acids revealed the location of the DNA template, the RNA product, and the NTP substrate, as well as elucidated many aspects of the transcription mechanism. 3. Structural studies of Pol II complexes with protein factors provided insights into transcription initiation and characterized the enzyme’s tunable active site, which catalyzes RNA synthesis and cleavage. 4. Recent structural studies of Pol I and Pol III revealed structural differences between the three eukaryotic RNA polymerases that account for functional differences, and enabled a detailed comparative structure-function analysis.
FUTURE ISSUES 1. What is the mechanism of nucleic acid translocation over the polymerase surface? How can structural and functional data on the nucleotide addition cycle be combined into a movie of the transcribing polymerase? 2. How can the crystal structures of Pol I and Pol III be determined to elucidate the detailed structural differences between eukaryotic RNA polymerases and to explain enzyme-specific properties and regulation? 3. What is the three-dimensional structure of the Pol II transcription initiation complex, and what does this tell us about promoter recognition, loading, and melting? 4. What are the mechanisms of transcriptional regulation? How do transcription factors change the amount of RNAs produced by the polymerases?
DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS We apologize to the many colleagues whose work was not mentioned because of the special focus of this review on structural studies of eukaryotic RNA polymerases and because of space restraints. This work was supported by the Deutsche Forschungsgemeinschaft, the Nanosystems Initiative Munich (NIM), the EU Network 3D Repertoire, the Elitenetzwerk Bayern,
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the Boehringer-Ingelheim Fonds, the EU Marie-Curie training program, and the Fonds der Chemischen Industrie.
NOTE ADDED IN PROOF
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While this review was in production the first crystal structure of an archaeal RNA polymerase became available (Hirata A, Klein BJ, Murakami KS. 2008. The X-ray crystal structure of RNA polymerase from archaea. Nature 451:851–54). This work confirmed the conclusions from a cryo-EM structure of another archaeal RNA polymerase (49) and provided many additional structural details.
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Contents
Annual Review of Biophysics Volume 37, 2008
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Frontispiece Robert L. Baldwin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p xiv The Search for Folding Intermediates and the Mechanism of Protein Folding Robert L. Baldwin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 How Translocons Select Transmembrane Helices Stephen H. White and Gunnar von Heijne p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 23 Unique Rotary ATP Synthase and Its Biological Diversity Christoph von Ballmoos, Gregory M. Cook, and Peter Dimroth p p p p p p p p p p p p p p p p p p p p p p p p 43 Mediation, Modulation, and Consequences of Membrane-Cytoskeleton Interactions Gary J. Doherty and Harvey T. McMahon p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 65 Metal Binding Affinity and Selectivity in Metalloproteins: Insights from Computational Studies Todor Dudev and Carmay Lim p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 97 Riboswitches: Emerging Themes in RNA Structure and Function Rebecca K. Montange and Robert T. Batey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p117 Calorimetry and Thermodynamics in Drug Design Jonathan B. Chaires p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p135 Protein Design by Directed Evolution Christian Jäckel, Peter Kast, and Donald Hilvert p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p153 PIP2 Is A Necessary Cofactor for Ion Channel Function: How and Why? Byung-Chang Suh and Bertil Hille p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p175 RNA Folding: Conformational Statistics, Folding Kinetics, and Ion Electrostatics Shi-Jie Chen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p197 Intrinsically Disordered Proteins in Human Diseases: Introducing the D2 Concept Vladimir N. Uversky, Christopher J. Oldfield, and A. Keith Dunker p p p p p p p p p p p p p p p p215 Crowding Effects on Diffusion in Solutions and Cells James A. Dix and A.S. Verkman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p247
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Nanobiotechnology and Cell Biology: Micro- and Nanofabricated Surfaces to Investigate Receptor-Mediated Signaling Alexis J. Torres, Min Wu, David Holowka, and Barbara Baird p p p p p p p p p p p p p p p p p p p p p p265 The Protein Folding Problem Ken A. Dill, S. Banu Ozkan, M. Scott Shell, and Thomas R. Weikl p p p p p p p p p p p p p p p p p p289 Translocation and Unwinding Mechanisms of RNA and DNA Helicases Anna Marie Pyle p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p317
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Structure of Eukaryotic RNA Polymerases P. Cramer, K.-J. Armache, S. Baumli, S. Benkert, F. Brueckner, C. Buchen, G.E. Damsma, S. Dengl, S.R. Geiger, A.J. Jasiak, A. Jawhari, S. Jennebach, T. Kamenski, H. Kettenberger, C.-D. Kuhn, E. Lehmann, K. Leike, J.F. Sydow, and A. Vannini p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p337 Structure-Based View of Epidermal Growth Factor Receptor Regulation Kathryn M. Ferguson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p353 Macromolecular Crowding and Confinement: Biochemical, Biophysical, and Potential Physiological Consequences Huan-Xiang Zhou, Germán Rivas, and Allen P. Minton p p p p p p p p p p p p p p p p p p p p p p p p p p p p p375 Biophysics of Catch Bonds Wendy E. Thomas, Viola Vogel, and Evgeni Sokurenko p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p399 Single-Molecule Approach to Molecular Biology in Living Bacterial Cells X. Sunney Xie, Paul J. Choi, Gene-Wei Li, Nam Ki Lee, and Giuseppe Lia p p p p p p p p p417 Structural Principles from Large RNAs Stephen R. Holbrook p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p445 Bimolecular Fluorescence Complementation (BiFC) Analysis as a Probe of Protein Interactions in Living Cells Tom K. Kerppola p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p465 Multiple Routes and Structural Heterogeneity in Protein Folding Jayant B. Udgaonkar p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p489 Index Cumulative Index of Contributing Authors, Volumes 33–37 p p p p p p p p p p p p p p p p p p p p p p p p511 Errata An online log of corrections to Annual Review of Biophysics articles may be found at http://biophys.annualreviews.org/errata.shtml viii
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