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RPB7 Complex
Molecular Cell, Vol. 8, 1137–1143, November, 2001, Copyright 2001 by Cell Press
Structure of an Archaeal Homolog of the Eukaryotic RNA Polymerase II RPB4/RPB7 Complex Flavia Todone, Peter Brick, Finn Werner, Robert O.J. Weinzierl, and Silvia Onesti1 Department of Biological Sciences Imperial College Exhibition Road London SW7 2AZ United Kingdom
Summary The eukaryotic subunits RPB4 and RPB7 form a heterodimer that reversibly associates with the RNA polymerase II core and constitute the only two components of the enzyme for which no structural information is available. We have determined the crystal structure of the complex between the Methanococcus jannaschii subunits E and F, the archaeal homologs of RPB7 and RPB4. Subunit E has an elongated twodomain structure and contains two potential RNA binding motifs, while the smaller F subunit wraps around one side of subunit E, at the interface between the two domains. We propose a model for the interaction between RPB4/RPB7 and the core RNA polymerase in which the RNA binding face of RPB7 is positioned to interact with the nascent RNA transcript. Introduction The transcription of DNA into RNA is carried out by RNA polymerases (RNAPs), large multisubunit enzymes containing a catalytic center and an assembly platform conserved from prokaryotes to humans. The best-studied eukaryotic system is the RNAPII from Saccharomyces cerevisiae, which includes 12 subunits (RPB112), of which two (RPB4 and RPB7) form a dissociable complex. RPB7 is an essential gene in yeast and its deletion is lethal (McKune et al., 1993; Young, 1991). In contrast, RPB4 is dispensable under optimal growth conditions: in rich medium at mild temperatures, cells lacking RPB4 (rpb4⌬) grow similarly to their wild-type counterparts and show almost normal transcriptional activity. However, whereas wild-type cells can grow over a wide range of temperatures, rpb4⌬ cells are heat and cold sensitive (Rosenheck and Choder, 1998; Woychik and Young, 1989). Moreover, cells lacking RPB4 are not able to enter the stationary phase when encountering nutrient depletion and die (Choder, 1993). RPB4 and RPB7 form a heterodimer that reversibly associates with the RNAPII 10 subunit core. The stoichiometry of RNAPII purified from S. cerevisiae cells is dependent on growth conditions: in exponentially growing cells only about 20% of purified enzyme includes the RPB4/RPB7 complex, while in the stationary phase the majority of polymerase molecules contain all 12 sub1
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units. RNAPII purified from the rpb4⌬ strain lacks both RPB4 and RPB7 (Edwards et al., 1991). These results have led researchers to conclude that the interaction between RPB7 and the RNAPII core is mediated by RPB4 and that the heterodimer plays a role in the transcription process only during stress. Yet, the fact that RPB7 is absolutely essential during optimal growth conditions is not consistent with this conclusion, and more recent results favor a model in which RPB7 is capable of interacting with RNAPII independently of RPB4 (Sheffer et al., 1999). RPB4 and RPB7 have also been implicated in promoter-specific transcription initiation in vitro (Edwards et al., 1991; Orlicky et al., 2001) and, more recently, in activated transcription in vivo (Pillai et al., 2001). No obvious homolog of RPB4 is found in either RNAPI or RNAPIII, while a polypeptide (C25) with significant sequence homology to RPB7 is found in RNAPIII (Sadhale and Woychik, 1994). All the RPB7 homologs contain a sequence motif called the S1 motif, often found in proteins involved in ssRNA binding. Gel mobility shift assays have shown that the yeast RPB4/RPB7 heterodimer binds to both ssDNA and ssRNA (Orlicky et al., 2001). The three-dimensional structure of the S. cerevisiae RNAPII has been determined at atomic resolution (Cramer et al., 2001; Gnatt et al., 2001). Since the heterogeneity in the polymerase preparations caused by the variable stoichiometry of the RPB4/RPB7 heterodimer interfered with crystallization, the enzyme used for the crystallographic studies was purified from the rpb4⌬ yeast strain, and therefore lacked both RPB4 and RPB7. As a consequence, the RPB4/RPB7 complex remains the only part of the RNA polymerase for which no structural model is currently available. Archaeal cells contain a single RNAP made up of about 12 subunits, displaying considerable homology to the eukaryotic RNAPII subunits. The RPB4 and RPB7 homologs are called subunits F and E, respectively, and have been shown to form a stable heterodimer (Werner et al., 2000). While the RPB7 homolog is reasonably well conserved (Figure 1), the similarity between the eukaryotic RPB4 and the archaeal F subunit is barely detectable. We have determined the crystal structure of the complex between the Methanococcus jannaschii subunits E and F to 1.75 A˚ resolution. The structure reveals the details of the interactions between the two subunits and sheds light on some of the biochemical data on the RPB4/RPB7 complex, including binding to single-stranded nucleic acids. We propose a mode of interaction between RPB4/RPB7 and the RNAP core and discuss the role of these two subunits in transcription. Results Overall Structure The crystal structure of the archaeal E/F complex from M. jannaschii was determined at 1.75 A˚ resolution by multiple isomorphous replacement and refined to an
Molecular Cell 1138
Figure 1. Amino Acid Sequence Alignment for the RPB7 Homologs An alignment of the Methanococcus jannaschii (Metja) E subunit with the eukaryotic Saccharomyces cerevisiae (Yeast) and the human (Human) RPB7 subunits. Highlighted in magenta are the residues that are conserved in at least 15 of the 21 available archaeal and eukaryotic sequences; highlighted in green are the residues conserved in 9/12 archaeal homologs; and highlighted in yellow are the amino acids conserved in 7/9 eukaryotic sequences (the following couples of amino acid residues have been considered similar: Phe/Tyr, Asp/Glu; Ser/Thr, Arg/Lys). The position of the secondary structure elements in the E subunit is shown above the sequence. A dashed line indicates residues omitted from the final model.
R-factor of 21.7 % (Table 1). The larger E subunit contains 187 amino acid residues and folds into two domains forming an elongated structure (Figure 2). The F subunit is smaller (107 residues) and folds into a number of ␣ helices that pack against one side of subunit E, at the hinge between the N-terminal and the C-terminal domains. The interface between E and F is dominated by hydrogen bonds and several salt bridges, rather than hydrophobic interactions. The electrostatic potential on the surface of the heterodimer shows a very unequal charge distribution (Figure 3): the “front” face of the E subunit is either neutral or positively charged, while the “back” of the complex, particularly the external surface of the F subunit, is highly negatively charged. The C-Terminal Domain of Subunit E Contains an S1 Motif The S1 homology domain has been named after the bacterial ribosomal protein S1, which contains six cop-
ies of a motif of approximately 70–80 amino acids (Subramanian, 1983). The same motif is found in a number of other proteins, some of which have been shown to bind single-stranded RNA in vitro. Despite the high degree of sequence similarity, no residue is absolutely conserved throughout the entire motif. The NMR structures of the S1 domain of the E. coli polynucleotide phosphorylase (Bycroft et al., 1997) and of transcription factor NusA (Worbs et al., 2001) reveal that the S1 motif folds into a five-stranded antiparallel  barrel, known as an OB fold. This is a common and ubiquitous structural domain, with no conserved sequence pattern, which is often involved in nucleic acid binding. The OB fold of the E subunit possesses many elements of the typical S1 motif but also displays a number of unusual features. The most striking difference is a long insertion between strands B3 and B4, where a short 310 helix (which is present in all the S1 domains) is followed by a three-stranded antiparallel  sheet (C1-C3).
Table 1. Data Collection and Phasing Statistics Native
PCMB (sat. sol.)
K2[PtBr4] (0.2 mM)
K[AuCl4] (0.2 mM)
28-1.75 76,814 3.6 99.8 (99.6) 9.2 (23.4) 4.1 (3.0)
20-2.5 26,400 3.4 99.4 (99.4) 9.0 (11.7) 4.9 (5.4)
20-2.8 21,085 3.3 99.4 (99.4) 7.8 (25.9) 6.5 (2.8)
20-2.5 26,146 3.3 98.3 (98.3) 9.9 (13.2) 4.5 (4.5)
0.64/0.70 1.20/1.57
0.65/0.65 1.36/1.74
0.82/0.88 0.58/1.04
Data Collection Resolution (A˚) Unique reflections Multiplicity Completeness (%)a Rmerge (%)b Intensity/a Phasing, 10-3 A˚ RCullis, centric/acentricc Phasing power, centric/acentric Mean figure of merit a
0.46
Values in parentheses refer to the highest resolution bin. b Rmerge ⫽ ⌺h⌺I|Ii(h) ⫺ 具I(h)典|/⌺h⌺iIi(h), where Ii(h) is the ith measurement of reflection h and 具I(h)典 is the weighted mean of all measurements of I(h). c RCullis is defined as the isomorphous lack of closure over the isomorphous difference.
Structure of an Archaeal RPB4/RPB7 Complex 1139
Figure 2. The Overall Structure of the E/F Complex (A) Stereo diagram showing a ribbon representation of the heterodimer. The E subunit (shown in blue) is an elongated molecule that folds into two domains: a  sheet (A) wrapped around a helix (K2) forms the N-terminal domain while the S1 motif in the C-terminal half of the protein folds into an antiparallel  barrel (B) with an OB-fold topology. The secondary structure elements that are part of the canonical S1 motif are shown in light blue. The disordered loop (residues 152 to 158) between strands B4 and B5 in the OB fold has been modeled for the sake of clarity and is shown as an orange dashed line. The F subunit (shown in magenta) folds into a series of helices that pack against one side of the E subunit at the interface between the two domains, with the N terminus contributing one strand to the N-terminal  sheet of E. (B) Schematic diagram showing the topology of the E/F complex. The secondary elements are colored and labeled as in (A).
Although the sequence conservation in this region is low (Figure 1), some features suggest that such a structure is likely to be also present in the eukaryotic RPB7 and C25 family: the length of the insertion between strands B3 and B4 is similar, and some of the residues with a structural role (Asp 116 and Tyr 120) are conserved. Another unusual feature is a large “bulge” that interrupts the fifth strand of the barrel (breaking it into B5 and B6) and forms a groove that accommodates one helix of subunit F. An additional ␣ helix (K4), located after the  barrel, is conserved only in the archaeal sequences and is probably a specialized feature of the prokaryotic subunit. A cluster of conserved surface-exposed amino acids is located on one side of the  barrel and includes charged or polar residues (Asp 105, His 109, Ser 111, Gln 112, and Lys 160) and aromatic residues (Phe 95 and Phe 98). All these amino acids are well conserved in archaea, but only Phe 98 is also present in most
eukaryotic homologs (Figure 1). On the other hand, a similar pattern of exposed, well-conserved residues for the eukaryotic RPB7 subunits can be inferred from the structure of the archaeal E subunit. To date, no direct structural information is available on the interaction of an S1 domain with nucleic acids, but the structures of several proteins containing OB folds have been determined in the presence of RNA or DNA. Although the specific details of the interaction differ, in all cases oligonucleotide binding occurs on the face of the barrel corresponding to that containing the conserved residues in the E subunit. In both copies of the E subunit present in the asymmetric unit, no electron density is visible for part of the loop connecting strand B4 and B5. In a number of OB folds, this loop has been shown to play a central role in DNA or RNA binding and often undergoes a conformational change to trap the nucleic acids.
Molecular Cell 1140
Figure 3. Surface Electrostatic Potential Mapping of the electrostatic potential on the surface of the E/F complex, with negative charges shown in red and positive charges in blue. On the left-hand side of the picture, the complex is shown in the same orientation as in Figure 2A (front), while on the right-hand side it is rotated by 180⬚ around a vertical axis (back). The heterodimer shows a strikingly asymmetric charge distribution, with one side of the complex (particularly subunit F) highly negatively charged.
The N-Terminal Domain of Subunit E Contains a Truncated RNP Motif The topology of the N-terminal domain of subunit E resembles a truncated RNP fold. RNP domains are single-stranded RNA binding modules of about 80 amino acid residues, which fold into a 1-␣1-2-3-␣2-4 structure, with the four antiparallel  strands packing against the two helices (Varani and Nagai, 1998). Canonical RNP domains can be identified by the presence of two sequence motifs (RNP1 and RNP2), which are located on the two central  strands (3 and 1, respectively) and contain conserved charged and aromatic side chains involved in interactions with the RNA. A database search with the N-terminal domain of the E subunit, using the program DALI (Holm and Sander, 1993), revealed a structural similarity with the B8 domain of phenylalanine tRNA synthetase (PheRS-B8, Goldgur et al., 1997) and with ribosomal protein S6 (Lindahl et al., 1994), which both have the characteristic topology of an RNP fold but lack the RNP1 and RNP2 sequence motifs. When the N-terminal domain of subunit E is superposed onto S6 and PheRS-B8, the secondary structure elements A1, K2, A3, and A4 (Figure 2B) overlap with 1, ␣1, 2, and 3 of the RNP motif. Helix ␣2 and strand 4 are missing in subunit E, although the strand contributed by the F subunit (A1⬘) topologically replaces strand 4, at least in part. The similarity is more pronounced in the case of PheRS-B8, which also shows some sequence similarity (19% of sequence identity between structurally equivalent residues) to subunit E. Both domains are more elongated than the standard RNP fold and display a similar twist in the  sheet. The loop between strands A3 and A4 is equivalent to the 2-3 loop of a RNP domain, which is often flexible and becomes more ordered upon RNA binding. In the structure of the E subunit, the region between residues 58 and 65 (which includes the loop A3-A4) displays temperature factors that are considerably higher than average and has a different conformation in the two crystallographically independent molecules. In the complex between PheRS and the cognate tRNAPhe, this loop is tightly involved in interaction with nucleic acid. However,
the recognition of the tRNA anticodon by PheRS-B8 is rather different from the RNA recognition in other RNP motifs. The structural and sequence similarity between the PheRS anticodon binding domain and the E subunit suggests that the N-terminal domain may also be involved in nucleic acid binding. The residues shown to interact with ribonucleotides in PheRS-B8 are different in the archaeal E sequences, but this is not very surprising since the role of the synthetase residues is to ensure specificity for the correct anticodon. The fact that the bottom part of the N-terminal domain (Figure 2), roughly equivalent to the anticodon binding region of PheRS, is well conserved in archaea and includes a few solventexposed residues (Lys 12, Phe 18, Glu 56, Gly 62, Asp 63, and Tyr 67), suggests a possible role in nucleic acid binding. This conserved patch continues over the bottom of the molecule toward the back with a large cluster of small surface-exposed residues (Pro 15, Gly 55, Gly 57, Gly 62, and Gly 64) highly conserved in both the archaeal and eukaryotic families (Figure 1). However, it is not clear whether the high level of conservation of this region is due to a role in the interactions with the RNAP core or nucleic acid binding. The F Subunit Binds to Subunit E at the Domain Interface The N-terminal residues of the F subunit contribute an extra strand (A1⬘) to the N-terminal  sheet of E (Figure 2B); many instances of similar “ addition motifs” have been described in the RNAPII architecture (Cramer et al., 2001). The remainder of the polypeptide chain folds into six ␣ helices, forming a semicircular “belt” that wraps around one side of subunit E, at the interface between the N-terminal and C-terminal domains (Figure 2). The overall sequence homology for this subunit is low: the similarity between the archaeal and eukaryotic homologs is barely detectable, and until recently it was thought that archaeal cells lacked a counterpart of the eukaryotic RPB4. Biochemical studies on the purified Methanococcus thermoautotrophicum RNAP (Darcy et
Structure of an Archaeal RPB4/RPB7 Complex 1141
al., 1999) and protein interaction studies of the M. jannaschii F and E subunits (Werner et al., 2000) have shown the existence of a true archaeal RPB4 homolog. Even within the eukaryotic RPB4 family the sequence homology is very low; in particular the S. cerevisiae RPB4 subunit is unusually large and is characterized by an N-terminal extension and a long insertion loop located between helices H1 and H2 in the F subunit. Apart from the residues involved in structural interactions (within F and at the E/F interface), no surface-exposed amino acids are conserved. A database search with the program DALI (Holm and Sander, 1993) identified a structural similarity between helices H3, H4, H5, and H6 with the C-terminal domain of RecQ helicases. This is a module of about 90 amino acids found in a number of RecQ helicases and RNaseD homologs from various organisms and named HRDC (helicase and RNaseD C-terminal) domain. The structure of this module from the S. cerevisiae RecQ helicase Sgs1 has been determined by NMR and has been shown to bind to ssDNA with low affinity and no sequence specificity (Liu et al., 1999). However, only one of the residues that interacts with DNA is conserved within the HRDC family itself, and the charge distribution on the surface of the module is not conserved, suggesting that this domain may simply represent a common structural motif in nucleic acid metabolism, with no conserved functional role. The fact that none of the residues that have been implicated in DNA binding in the Sgs1 HRDC domain is conserved and the electrostatic character of the surface is very different (positively charged in HRDC and negatively charged in subunit F) makes it unlikely that the surface of the F subunit is similarly involved in nucleic acid binding. Discussion A Model for the Interaction of the RPB4/RPB7 Complex with the RNAP Core When discussing the possible role of the RPB4/RPB7 heterodimer in transcription, an obvious problem that needs to be addressed is the location with respect to the 10 subunit RNAP core. Due to the lack of detailed biochemical and genetic information on the archaeal transcriptional machinery, most of the following discussion will focus on the data available for eukaryotic systems, and particularly for the S. cerevisiae RNAPII. A comparison of the atomic structures of yeast RNAPII in the absence (Cramer et al., 2001) and the presence (Gnatt et al., 2001) of nucleic acids shows a movement of a large domain (the “clamp”) that closes over the active site, trapping the DNA template and DNA/RNA hybrid, and suggests a putative exit groove (the “saddle”) for the nascent RNA transcript at the base of the clamp. Information from different sources can be used to infer the position and the orientation of the RPB4/RPB7 heterodimer. The most direct piece of evidence comes from a 24 A˚ electron diffraction difference map between the yeast 12 subunit RNAPII and the 10 subunit core lacking the RPB4/RPB7 heterodimer (Jensen et al., 1998). Although it is difficult to decouple the additional density from the conformational changes in the polymer-
ase, a peak of positive density indicates that RPB4/ RPB7 is located between RPB5 and the base of the clamp (Cramer et al., 2000). The surface of the RNAPII 10 subunit core is highly negatively charged (Cramer et al., 2001). Aside from the active site cleft, which binds the DNA template and the DNA/RNA hybrid, the only surfaces that are either neutral or positively charged are the saddle and the base of the clamp. The highly asymmetric charge distribution of the E/F heterodimer (Figure 3) suggests a likely orientation for the complex (Figure 4), with the F subunit on the outside, maintaining the overall negative charge on the external surface of the RNAP core. Any other alternative would place highly negative surfaces (the polymerase core and the back of the E/F dimer) in close contacts. An additional argument supports the proposed orientation: in the yeast homolog a long insertion loop is located between helices H1 and H2, suggesting that this side of the complex is unlikely to be in contact with the core. RPB7 Is Positioned to Interact with the Nascent RNA Transcript The presence of an S1 motif in the sequence of the eukaryotic RPB7 and archaeal E subunit strongly suggests that the role of this subunit is to bind singlestranded nucleic acids. This has been shown in the case of the S. cerevisiae RPB4/RPB7 complex (Orlicky et al., 2001). Although a number of OB folds bind ssDNA as well as RNA, all the known members of the S1 subfamily bind to ssRNA, often in a sequence-nonspecific manner. In most of these proteins, RNA binding is spread over more than one domain, with the nucleic acid binding site forming a continuous surface. This is exemplified in the structure of NusA, which forms an extended RNA binding surface that spirals around the S1 motif and the KH domain (Worbs et al., 2001). If the truncated RNP fold in the N-terminal domain of subunit E does indeed bind nucleic acid, this subunit could contribute to a similar extended RNA binding surface. One argument in favor of this hypothesis is the fact that a partially conserved “path” can be identified along the surface of the molecule, leading from the S1 motif binding surface, through Lys 40, Asp 41, Arg 37, and Lys 3, down to the putative nucleic acid binding region close to the loop between strands A3 and A4. One interesting aspect of this path is the relatively high temperature factors displayed by the residues involved, suggesting that a certain degree of flexibility may be instrumental to nucleic acid binding. We favor a model in which the S1 motif of RPB7 interacts with the nascent RNA transcript, possibly assisted by the truncated RNP motif (Figure 4); this model is consistent with the predicted position of the heterodimer at the base of the clamp (Cramer et al., 2000), where the RNA exit groove is located. Apart from the emerging RNA transcript, the only other occurrence of single stranded nucleic acid in transcription is the noncoding DNA strand in the transcription bubble, which is likely to be almost entirely enclosed within the RNAP core. Moreover, this would require RPB4/RPB7 to be located in a position incompatible with the EM results. Furthermore, our model is in agreement with the observation that the RPB4/RPB7 heterodimer favors a
Molecular Cell 1142
Figure 4. Proposed Model for the Interaction of RPB4/RPB7 with the RNA Polymerase Core A schematic representation of the RNAPII 10 subunit core is shown (viewed in a similar orientation as the side view in Figures 3 and 6D of Cramer et al. (2000), with RPB5 shown in red and the mobile clamp, which closes onto the active side cleft upon nucleic acid binding, shown in green. The predicted exit path for the nascent RNA transcript is shown as a dashed line, and the proposed location of the heterodimer shown as a blue circle. The structure of the E/F complex is shown rotated by approximately 130⬚ around a vertical axis with respect to the orientation used in Figure 2A. The position and orientation of the heterodimer is consistent with a wide range of structural and biological results, including low-resolution studies on 2D crystals and the positioning of the S1 motif binding face onto the nascent RNA transcript. The negatively charged surface of the F subunit is positioned on the outside, the insertion in the yeast RPB4 structure can be easily accommodated, and RPB7 plays the main role in the interaction, in agreement with the biochemical and genetic results.
closed conformation of the polymerase around the DNA binding cleft and increases DNA binding (Jensen et al., 1998). Binding of RPB7 to the nascent transcript may further enhance the conformational changes triggered by the presence of the DNA/RNA hybrid, leading to a tightly bound and more stable transcribing complex. Indeed, a three-step model for transcription initiation has been proposed (Holstege et al., 1997), where the third step is a structural transition occurring after the synthesis of the first 10–11 ribonucleotides, accompanied by a switch from abortive to productive RNA synthesis and promoter clearance. Binding of the nascent transcript to RPB7 may be coupled to the movement of the clamp to help achieve this last conformational change leading to a stable transcribing complex. RPB4 Has a Stabilizing Role There have been a number of reports suggesting a specific role for the heterodimer, and particularly RPB4, in transcription during stress. However, no explicit correlation has ever been demonstrated between either RPB4 or RPB7 and the specific transcription of heat-shock or stress genes. Despite the normal growth rate, rpb4⌬ cells exhibit stress-unrelated transcriptional defects, showing that this subunit has a general role in efficient mRNA synthesis under normal growth; therefore the stress response anomalies may be secondary consequences of a general transcription defect and the consequent perturbation of cellular functions (Bourbonnais et al., 2000; Tan et al., 2000). It has also been shown that overexpression of RPB7 can partly suppress some of the rpb4⌬ phenotypes, and that this subunit can interact with the RNAPII in the absence of RPB4, albeit less efficiently, making RPB7 the critical component of the dimer (Sheffer et al., 1999). The role of RPB4 is likely to be to stabilize the conformation of RPB7. The structure of the E/F heterodimer
strongly argues in favor of this conclusion, since no particular feature seems to suggest a functional role for this subunit either in binding nucleic acid or interacting with the polymerase core. The F subunit packs against one side of E at the hinge between the N-terminal and C-terminal domains as a “protective belt.” RPB4 probably facilitates the recruitment of RPB7 to the polymerase core, by enhancing its stability and/or supplying additional contacts. Under stress, an efficient interaction between the polymerase core and RPB7 becomes crucial for cell viability and the stabilizing presence of RPB4 indispensable. Experimental Procedures Protein Expression and Purification Coexpression of a recombinant full-length M. jannaschii E/F complex was achieved using a bicistronic expression strategy in E. coli BL21 (DE3) cells using a pGEX-2TK vector (Pharmacia), with the F subunit produced as a fusion with glutathione S transferase (Werner et al., 2000). For crystallization experiments, the protein was equilibrated with crystallization buffer (20 mM MES [pH 6.5], 100 mM sodium chloride, and 10 mM -mercaptoethanol) and concentrated with a Centricon-10 micro-concentrator (Amicon) to a final protein concentration of 20–25 mg/ml. Crystallization and Data Collection Crystals were grown at 4⬚C by vapor diffusion in hanging drops, equilibrated against a well solution containing 1.3–1.6 M ammonium dihydrogen monophosphate and 100 mM Tris/HCl (pH 4.6). The diffraction pattern is consistent with the tetragonal space group P43 with cell dimensions of a ⫽ b ⫽ 92 A˚ and c ⫽ 91 A˚. A self-rotation function showed the presence of a noncrystallographic 2-fold axis relating two complexes in the asymmetric unit. All data were collected at 100 K at beamline ID14-4 of the European Synchrotron Radiation Facility (ESRF, Grenoble, France). The images were processed and integrated using MOSFLM and subsequently handled using the CCP4 package (Collaborative Computational Project Number 4, 1994). Data collection statistics are summarized in Table 1.
Structure of an Archaeal RPB4/RPB7 Complex 1143
Structure Solution and Refinement The structure was solved by the multiple isomorphous replacement method (Table 1), followed by solvent flattening and noncrystallographic symmetry averaging, and refined with the program CNS (Bru¨nger et al., 1998), using all reflections between 20 and 1.75 A˚. The final model has a crystallographic R-factor of 21.7% (Rfree⫽ 23.8% calculated using 5% of the data) with a bond length r.m.s. deviation from ideality of 0.006 A˚. The two complexes in the asymmetric unit are very similar and can be superposed with a root-mean-square deviation of 0.39 A˚ between equivalent ␣ carbons. Analysis of the model with PROCHECK (Laskowski et al., 1993) shows good geometry with all residues in the allowed regions of the Ramachandran plot. Acknowledgments This work was supported by a Wellcome Trust Grant to S.O, P.B., and R.O.J.W. We are very grateful to Gordon Leonard for assistance during data collection at ESRF. Figures 2, 3, and 4 were prepared using the programs MOLSCRIPT (Esnouf, 1997), RASTER3D (Merritt and Bacon, 1997), and GRASP (Nicholls et al., 1991).
Holm, L., and Sander, C. (1993). Protein structure comparison by alignment of distance matrices. J. Mol. Biol. 233, 123–138. Holstege, F.C.P., Fiedler, U., and Timmers, H.T.M. (1997). Three transitions in the RNA polymerase II transcription complex during initiation. EMBO J. 16, 7468–7480. Jensen, G.J., Merendith, G., Bushnell, D.A., and Kornberg, R.D. (1998). Structure of the wild-type yeast RNA polymerase II and location of Rpb4 and Rpb7. EMBO J. 17, 2353–2358. Laskowski, R.A., MacArthur, M.V., Moss, D.S., and Thornton, J.M. (1993). PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291. Lindahl, M., Svensson, L.A., Liljas, A., Sedelnikova, S.E., Eliseikina, I.A., Formenkova, N.P., Nevskaya, N., Nikonov, S.V., Garber, M.B., Muranova, T.A., et al. (1994). Crystal structure of the ribosomal protein S6 from Thermus thermophilus. EMBO J. 13, 1249–1254. Liu, Z., Macias, M.J., Bottomley, M.J., Sier, G., Linge, J.P., Nilges, M., Bork, P., and Sattler, M. (1999). The three-dimensional structure of the HRDC domain and implications for the Werner and Bloom syndrome proteins. Struct. Fold. Des. 7, 1557–1566.
Received July 23, 2001; revised September 26, 2001.
McKune, K., Richards, K.L., Edwards, A.M., Young, R.A., and Woychik, N.A. (1993). RPB7, one of two dissociable subunits of yeast RNA polymerase II, is essential for cell viability. Yeast 9, 295–299.
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Bourbonnais, Y., Faucher, N., Pallotta, D., and Larouche, C. (2000). Multiple cellular processes affected by the absence of the Rpb4 subunit of RNA polymerase II contribute to the deficiency in the stress response of the yeast rpb4⌬ mutant. Mol. Gen. Genet. 264, 763–772. Bru¨nger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. (1998). Crystallographic and NMR system: a new software system for macromolecular structure determination. Acta Crystallogr. D 50, 760–763. Bycroft, M., Hubbard, T.J.P., Proctor, M., Freund, S.M.V., and Murzin, A.G. (1997). The solution structure of the S1 RNA binding domain: a member of an ancient nucleic acid-binding family. Cell 88, 235–242. Choder, M. (1993). A growth rate-limiting process in the last growth phase of the yeast life-cycle involves RPB4, a subunit of RNA polymerase II. J. Bacteriol. 175, 6358–6363. CCP4 (Collaborative Computational Project Number 4) (1994). The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763. Cramer, P., Bushnell, D.A., Fu, J., Gnatt, A.L., Maier-Davis, B., Thompson, N.E., Burgess, R.R., Edwards, A.M., David, P.R., and Kornberg, R.D. (2000). Architecture of RNA polymerase II and implication for the transcription mechanism. Science 288, 640–649.
Nicholls, A., Sharp, K.A., and Honig, B. (1991). Protein folding and association: insights from the interfacial and thermodynamics properties of hydrocarbons. Proteins 11, 281–296. Orlicky, S.M., Trans, P.T., Sayre, M.H., and Edwards, A.M. (2001). Dissociable Rpb4-Rpb7 subassembly of RNA polymerase II binds to single-stranded nucleic acid and mediates a post-recruitment step in transcription initiation. J. Biol. Chem. 276, 10097–10102. Pillai, B., Sampath, V., Sharma, N., and Sadhale, P. (2001). Rpb4, a non essential subunit of core RNA polymerase II of Saccharomyces cerevisiae is important for activate transcription of a subset of genes. J. Biol. Chem. 276, 30641–30647. Rosenheck, S., and Choder, M. (1998). Rpb4, a subunit of RNA polymerase II, enables the enzyme to transcribe at temperature extremes in vitro. J. Bacteriol. 180, 6187–6192. Sadhale, P.P., and Woychik, N.A. (1994). C25, an essential RNA polymerase III subunit related to the RNA polymerase II subunit RPB7. Mol. Cell. Biol. 14, 6164–6170. Sheffer, A., Varon, M., and Choder, M. (1999). Rpb7 can interact with RNA polymerase II and support transcription during some stresses independently of Rpb4. Mol. Cell. Biol. 19, 2672–2680. Subramanian, A.R. (1983). Structure and functions of ribosomal protein S1. Prog. Nucleic Acid Res. Mol. Biol. 28, 101–142. Tan, Q., Li, X., Sadhale, P.P., Miyao, T., and Woychik, N. (2000). Multiple mechanisms of suppression circumvent transcription defects in RNA polymerase mutant. Mol. Cell. Biol. 20, 8124–8133.
Cramer, P., Bushnell, D.A., and Kornberg, R.D. (2001). Structural basis of transcription: RNA polymerase II at 2.8 A˚ resolution. Science 292, 1863–1876.
Varani, G., and Nagai, K. (1998). RNA recognition by RNP proteins during RNA processing. Annu. Rev. Biophys. Biomol. Struct. 27, 407–445.
Darcy, T.J., Hausner, W., Awery, D.E., Edwards, A.M., Thomm, M., and Reeve, J.N. (1999). Methanococcus thermoautotrophicum RNA polymerase and transcription in vitro. J. Bacteriol. 181, 4424–4429.
Werner, F., Eloranta, J.J., and Weinzierl, R.O.J. (2000). Archaeal RNA polymerase subunits F and P are bona fide homologs of eukaryotic RPB4 and RPB12. Nucleic Acids Res. 28, 4299–4305.
Edwards, A.M., Kane, C.M., Young, R.A., and Kornberg, R.D. (1991). Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro. J. Biol. Chem. 266, 71–75.
Worbs, M., Bourenkov, G.P., Bartunik, H.D., Huber, R., and Wahl, M.C. (2001). An extended RNA binding surface through arrayed S1 and KH domains in transcription factor NusA. Mol. Cell 7, 1177–1189.
Esnouf, R.M. (1997). An extensively modified version of Molscript that includes greatly enhanced coloring capabilities. J. Mol. Graphics 15, 133–138. 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 3.3 A˚ resolution. Science 292, 1876–1882. Goldgur, Y., Mosyak, L., Reshetnikova, L., Ankilova, V., Lavrik, O., Khodyreva, S., and Safro, M. (1997). The crystal structure of phenylalanyl-tRNA synthetase from Thermus thermophilus complexed with cognate tRNAPhe. Structure 5, 59–68.
Woychik, N.A., and Young, R.A. (1989). RNA polymerase II subunit RPB4 is essential for high- and low- temperature yeast cell growth. Mol. Cell. Biol. 9, 2854–2859. Young, R.A. (1991). RNA polymerase II. Annu. Rev. Biochem. 60, 689–715. Accession Numbers Coordinates for the EF complex are in the Protein Data Bank under accession number 1go3.
Structure of an Archaeal Homolog of the Eukaryotic RNA Polymerase II RPB4/RPB7 Complex Flavia Todone, Peter Brick, Finn Werner, Robert O.J. Weinzierl, and Silvia Onesti1 Department of Biological Sciences Imperial College Exhibition Road London SW7 2AZ United Kingdom
Summary The eukaryotic subunits RPB4 and RPB7 form a heterodimer that reversibly associates with the RNA polymerase II core and constitute the only two components of the enzyme for which no structural information is available. We have determined the crystal structure of the complex between the Methanococcus jannaschii subunits E and F, the archaeal homologs of RPB7 and RPB4. Subunit E has an elongated twodomain structure and contains two potential RNA binding motifs, while the smaller F subunit wraps around one side of subunit E, at the interface between the two domains. We propose a model for the interaction between RPB4/RPB7 and the core RNA polymerase in which the RNA binding face of RPB7 is positioned to interact with the nascent RNA transcript. Introduction The transcription of DNA into RNA is carried out by RNA polymerases (RNAPs), large multisubunit enzymes containing a catalytic center and an assembly platform conserved from prokaryotes to humans. The best-studied eukaryotic system is the RNAPII from Saccharomyces cerevisiae, which includes 12 subunits (RPB112), of which two (RPB4 and RPB7) form a dissociable complex. RPB7 is an essential gene in yeast and its deletion is lethal (McKune et al., 1993; Young, 1991). In contrast, RPB4 is dispensable under optimal growth conditions: in rich medium at mild temperatures, cells lacking RPB4 (rpb4⌬) grow similarly to their wild-type counterparts and show almost normal transcriptional activity. However, whereas wild-type cells can grow over a wide range of temperatures, rpb4⌬ cells are heat and cold sensitive (Rosenheck and Choder, 1998; Woychik and Young, 1989). Moreover, cells lacking RPB4 are not able to enter the stationary phase when encountering nutrient depletion and die (Choder, 1993). RPB4 and RPB7 form a heterodimer that reversibly associates with the RNAPII 10 subunit core. The stoichiometry of RNAPII purified from S. cerevisiae cells is dependent on growth conditions: in exponentially growing cells only about 20% of purified enzyme includes the RPB4/RPB7 complex, while in the stationary phase the majority of polymerase molecules contain all 12 sub1
Correspondence: [email protected]
units. RNAPII purified from the rpb4⌬ strain lacks both RPB4 and RPB7 (Edwards et al., 1991). These results have led researchers to conclude that the interaction between RPB7 and the RNAPII core is mediated by RPB4 and that the heterodimer plays a role in the transcription process only during stress. Yet, the fact that RPB7 is absolutely essential during optimal growth conditions is not consistent with this conclusion, and more recent results favor a model in which RPB7 is capable of interacting with RNAPII independently of RPB4 (Sheffer et al., 1999). RPB4 and RPB7 have also been implicated in promoter-specific transcription initiation in vitro (Edwards et al., 1991; Orlicky et al., 2001) and, more recently, in activated transcription in vivo (Pillai et al., 2001). No obvious homolog of RPB4 is found in either RNAPI or RNAPIII, while a polypeptide (C25) with significant sequence homology to RPB7 is found in RNAPIII (Sadhale and Woychik, 1994). All the RPB7 homologs contain a sequence motif called the S1 motif, often found in proteins involved in ssRNA binding. Gel mobility shift assays have shown that the yeast RPB4/RPB7 heterodimer binds to both ssDNA and ssRNA (Orlicky et al., 2001). The three-dimensional structure of the S. cerevisiae RNAPII has been determined at atomic resolution (Cramer et al., 2001; Gnatt et al., 2001). Since the heterogeneity in the polymerase preparations caused by the variable stoichiometry of the RPB4/RPB7 heterodimer interfered with crystallization, the enzyme used for the crystallographic studies was purified from the rpb4⌬ yeast strain, and therefore lacked both RPB4 and RPB7. As a consequence, the RPB4/RPB7 complex remains the only part of the RNA polymerase for which no structural model is currently available. Archaeal cells contain a single RNAP made up of about 12 subunits, displaying considerable homology to the eukaryotic RNAPII subunits. The RPB4 and RPB7 homologs are called subunits F and E, respectively, and have been shown to form a stable heterodimer (Werner et al., 2000). While the RPB7 homolog is reasonably well conserved (Figure 1), the similarity between the eukaryotic RPB4 and the archaeal F subunit is barely detectable. We have determined the crystal structure of the complex between the Methanococcus jannaschii subunits E and F to 1.75 A˚ resolution. The structure reveals the details of the interactions between the two subunits and sheds light on some of the biochemical data on the RPB4/RPB7 complex, including binding to single-stranded nucleic acids. We propose a mode of interaction between RPB4/RPB7 and the RNAP core and discuss the role of these two subunits in transcription. Results Overall Structure The crystal structure of the archaeal E/F complex from M. jannaschii was determined at 1.75 A˚ resolution by multiple isomorphous replacement and refined to an
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Figure 1. Amino Acid Sequence Alignment for the RPB7 Homologs An alignment of the Methanococcus jannaschii (Metja) E subunit with the eukaryotic Saccharomyces cerevisiae (Yeast) and the human (Human) RPB7 subunits. Highlighted in magenta are the residues that are conserved in at least 15 of the 21 available archaeal and eukaryotic sequences; highlighted in green are the residues conserved in 9/12 archaeal homologs; and highlighted in yellow are the amino acids conserved in 7/9 eukaryotic sequences (the following couples of amino acid residues have been considered similar: Phe/Tyr, Asp/Glu; Ser/Thr, Arg/Lys). The position of the secondary structure elements in the E subunit is shown above the sequence. A dashed line indicates residues omitted from the final model.
R-factor of 21.7 % (Table 1). The larger E subunit contains 187 amino acid residues and folds into two domains forming an elongated structure (Figure 2). The F subunit is smaller (107 residues) and folds into a number of ␣ helices that pack against one side of subunit E, at the hinge between the N-terminal and the C-terminal domains. The interface between E and F is dominated by hydrogen bonds and several salt bridges, rather than hydrophobic interactions. The electrostatic potential on the surface of the heterodimer shows a very unequal charge distribution (Figure 3): the “front” face of the E subunit is either neutral or positively charged, while the “back” of the complex, particularly the external surface of the F subunit, is highly negatively charged. The C-Terminal Domain of Subunit E Contains an S1 Motif The S1 homology domain has been named after the bacterial ribosomal protein S1, which contains six cop-
ies of a motif of approximately 70–80 amino acids (Subramanian, 1983). The same motif is found in a number of other proteins, some of which have been shown to bind single-stranded RNA in vitro. Despite the high degree of sequence similarity, no residue is absolutely conserved throughout the entire motif. The NMR structures of the S1 domain of the E. coli polynucleotide phosphorylase (Bycroft et al., 1997) and of transcription factor NusA (Worbs et al., 2001) reveal that the S1 motif folds into a five-stranded antiparallel  barrel, known as an OB fold. This is a common and ubiquitous structural domain, with no conserved sequence pattern, which is often involved in nucleic acid binding. The OB fold of the E subunit possesses many elements of the typical S1 motif but also displays a number of unusual features. The most striking difference is a long insertion between strands B3 and B4, where a short 310 helix (which is present in all the S1 domains) is followed by a three-stranded antiparallel  sheet (C1-C3).
Table 1. Data Collection and Phasing Statistics Native
PCMB (sat. sol.)
K2[PtBr4] (0.2 mM)
K[AuCl4] (0.2 mM)
28-1.75 76,814 3.6 99.8 (99.6) 9.2 (23.4) 4.1 (3.0)
20-2.5 26,400 3.4 99.4 (99.4) 9.0 (11.7) 4.9 (5.4)
20-2.8 21,085 3.3 99.4 (99.4) 7.8 (25.9) 6.5 (2.8)
20-2.5 26,146 3.3 98.3 (98.3) 9.9 (13.2) 4.5 (4.5)
0.64/0.70 1.20/1.57
0.65/0.65 1.36/1.74
0.82/0.88 0.58/1.04
Data Collection Resolution (A˚) Unique reflections Multiplicity Completeness (%)a Rmerge (%)b Intensity/a Phasing, 10-3 A˚ RCullis, centric/acentricc Phasing power, centric/acentric Mean figure of merit a
0.46
Values in parentheses refer to the highest resolution bin. b Rmerge ⫽ ⌺h⌺I|Ii(h) ⫺ 具I(h)典|/⌺h⌺iIi(h), where Ii(h) is the ith measurement of reflection h and 具I(h)典 is the weighted mean of all measurements of I(h). c RCullis is defined as the isomorphous lack of closure over the isomorphous difference.
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Figure 2. The Overall Structure of the E/F Complex (A) Stereo diagram showing a ribbon representation of the heterodimer. The E subunit (shown in blue) is an elongated molecule that folds into two domains: a  sheet (A) wrapped around a helix (K2) forms the N-terminal domain while the S1 motif in the C-terminal half of the protein folds into an antiparallel  barrel (B) with an OB-fold topology. The secondary structure elements that are part of the canonical S1 motif are shown in light blue. The disordered loop (residues 152 to 158) between strands B4 and B5 in the OB fold has been modeled for the sake of clarity and is shown as an orange dashed line. The F subunit (shown in magenta) folds into a series of helices that pack against one side of the E subunit at the interface between the two domains, with the N terminus contributing one strand to the N-terminal  sheet of E. (B) Schematic diagram showing the topology of the E/F complex. The secondary elements are colored and labeled as in (A).
Although the sequence conservation in this region is low (Figure 1), some features suggest that such a structure is likely to be also present in the eukaryotic RPB7 and C25 family: the length of the insertion between strands B3 and B4 is similar, and some of the residues with a structural role (Asp 116 and Tyr 120) are conserved. Another unusual feature is a large “bulge” that interrupts the fifth strand of the barrel (breaking it into B5 and B6) and forms a groove that accommodates one helix of subunit F. An additional ␣ helix (K4), located after the  barrel, is conserved only in the archaeal sequences and is probably a specialized feature of the prokaryotic subunit. A cluster of conserved surface-exposed amino acids is located on one side of the  barrel and includes charged or polar residues (Asp 105, His 109, Ser 111, Gln 112, and Lys 160) and aromatic residues (Phe 95 and Phe 98). All these amino acids are well conserved in archaea, but only Phe 98 is also present in most
eukaryotic homologs (Figure 1). On the other hand, a similar pattern of exposed, well-conserved residues for the eukaryotic RPB7 subunits can be inferred from the structure of the archaeal E subunit. To date, no direct structural information is available on the interaction of an S1 domain with nucleic acids, but the structures of several proteins containing OB folds have been determined in the presence of RNA or DNA. Although the specific details of the interaction differ, in all cases oligonucleotide binding occurs on the face of the barrel corresponding to that containing the conserved residues in the E subunit. In both copies of the E subunit present in the asymmetric unit, no electron density is visible for part of the loop connecting strand B4 and B5. In a number of OB folds, this loop has been shown to play a central role in DNA or RNA binding and often undergoes a conformational change to trap the nucleic acids.
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Figure 3. Surface Electrostatic Potential Mapping of the electrostatic potential on the surface of the E/F complex, with negative charges shown in red and positive charges in blue. On the left-hand side of the picture, the complex is shown in the same orientation as in Figure 2A (front), while on the right-hand side it is rotated by 180⬚ around a vertical axis (back). The heterodimer shows a strikingly asymmetric charge distribution, with one side of the complex (particularly subunit F) highly negatively charged.
The N-Terminal Domain of Subunit E Contains a Truncated RNP Motif The topology of the N-terminal domain of subunit E resembles a truncated RNP fold. RNP domains are single-stranded RNA binding modules of about 80 amino acid residues, which fold into a 1-␣1-2-3-␣2-4 structure, with the four antiparallel  strands packing against the two helices (Varani and Nagai, 1998). Canonical RNP domains can be identified by the presence of two sequence motifs (RNP1 and RNP2), which are located on the two central  strands (3 and 1, respectively) and contain conserved charged and aromatic side chains involved in interactions with the RNA. A database search with the N-terminal domain of the E subunit, using the program DALI (Holm and Sander, 1993), revealed a structural similarity with the B8 domain of phenylalanine tRNA synthetase (PheRS-B8, Goldgur et al., 1997) and with ribosomal protein S6 (Lindahl et al., 1994), which both have the characteristic topology of an RNP fold but lack the RNP1 and RNP2 sequence motifs. When the N-terminal domain of subunit E is superposed onto S6 and PheRS-B8, the secondary structure elements A1, K2, A3, and A4 (Figure 2B) overlap with 1, ␣1, 2, and 3 of the RNP motif. Helix ␣2 and strand 4 are missing in subunit E, although the strand contributed by the F subunit (A1⬘) topologically replaces strand 4, at least in part. The similarity is more pronounced in the case of PheRS-B8, which also shows some sequence similarity (19% of sequence identity between structurally equivalent residues) to subunit E. Both domains are more elongated than the standard RNP fold and display a similar twist in the  sheet. The loop between strands A3 and A4 is equivalent to the 2-3 loop of a RNP domain, which is often flexible and becomes more ordered upon RNA binding. In the structure of the E subunit, the region between residues 58 and 65 (which includes the loop A3-A4) displays temperature factors that are considerably higher than average and has a different conformation in the two crystallographically independent molecules. In the complex between PheRS and the cognate tRNAPhe, this loop is tightly involved in interaction with nucleic acid. However,
the recognition of the tRNA anticodon by PheRS-B8 is rather different from the RNA recognition in other RNP motifs. The structural and sequence similarity between the PheRS anticodon binding domain and the E subunit suggests that the N-terminal domain may also be involved in nucleic acid binding. The residues shown to interact with ribonucleotides in PheRS-B8 are different in the archaeal E sequences, but this is not very surprising since the role of the synthetase residues is to ensure specificity for the correct anticodon. The fact that the bottom part of the N-terminal domain (Figure 2), roughly equivalent to the anticodon binding region of PheRS, is well conserved in archaea and includes a few solventexposed residues (Lys 12, Phe 18, Glu 56, Gly 62, Asp 63, and Tyr 67), suggests a possible role in nucleic acid binding. This conserved patch continues over the bottom of the molecule toward the back with a large cluster of small surface-exposed residues (Pro 15, Gly 55, Gly 57, Gly 62, and Gly 64) highly conserved in both the archaeal and eukaryotic families (Figure 1). However, it is not clear whether the high level of conservation of this region is due to a role in the interactions with the RNAP core or nucleic acid binding. The F Subunit Binds to Subunit E at the Domain Interface The N-terminal residues of the F subunit contribute an extra strand (A1⬘) to the N-terminal  sheet of E (Figure 2B); many instances of similar “ addition motifs” have been described in the RNAPII architecture (Cramer et al., 2001). The remainder of the polypeptide chain folds into six ␣ helices, forming a semicircular “belt” that wraps around one side of subunit E, at the interface between the N-terminal and C-terminal domains (Figure 2). The overall sequence homology for this subunit is low: the similarity between the archaeal and eukaryotic homologs is barely detectable, and until recently it was thought that archaeal cells lacked a counterpart of the eukaryotic RPB4. Biochemical studies on the purified Methanococcus thermoautotrophicum RNAP (Darcy et
Structure of an Archaeal RPB4/RPB7 Complex 1141
al., 1999) and protein interaction studies of the M. jannaschii F and E subunits (Werner et al., 2000) have shown the existence of a true archaeal RPB4 homolog. Even within the eukaryotic RPB4 family the sequence homology is very low; in particular the S. cerevisiae RPB4 subunit is unusually large and is characterized by an N-terminal extension and a long insertion loop located between helices H1 and H2 in the F subunit. Apart from the residues involved in structural interactions (within F and at the E/F interface), no surface-exposed amino acids are conserved. A database search with the program DALI (Holm and Sander, 1993) identified a structural similarity between helices H3, H4, H5, and H6 with the C-terminal domain of RecQ helicases. This is a module of about 90 amino acids found in a number of RecQ helicases and RNaseD homologs from various organisms and named HRDC (helicase and RNaseD C-terminal) domain. The structure of this module from the S. cerevisiae RecQ helicase Sgs1 has been determined by NMR and has been shown to bind to ssDNA with low affinity and no sequence specificity (Liu et al., 1999). However, only one of the residues that interacts with DNA is conserved within the HRDC family itself, and the charge distribution on the surface of the module is not conserved, suggesting that this domain may simply represent a common structural motif in nucleic acid metabolism, with no conserved functional role. The fact that none of the residues that have been implicated in DNA binding in the Sgs1 HRDC domain is conserved and the electrostatic character of the surface is very different (positively charged in HRDC and negatively charged in subunit F) makes it unlikely that the surface of the F subunit is similarly involved in nucleic acid binding. Discussion A Model for the Interaction of the RPB4/RPB7 Complex with the RNAP Core When discussing the possible role of the RPB4/RPB7 heterodimer in transcription, an obvious problem that needs to be addressed is the location with respect to the 10 subunit RNAP core. Due to the lack of detailed biochemical and genetic information on the archaeal transcriptional machinery, most of the following discussion will focus on the data available for eukaryotic systems, and particularly for the S. cerevisiae RNAPII. A comparison of the atomic structures of yeast RNAPII in the absence (Cramer et al., 2001) and the presence (Gnatt et al., 2001) of nucleic acids shows a movement of a large domain (the “clamp”) that closes over the active site, trapping the DNA template and DNA/RNA hybrid, and suggests a putative exit groove (the “saddle”) for the nascent RNA transcript at the base of the clamp. Information from different sources can be used to infer the position and the orientation of the RPB4/RPB7 heterodimer. The most direct piece of evidence comes from a 24 A˚ electron diffraction difference map between the yeast 12 subunit RNAPII and the 10 subunit core lacking the RPB4/RPB7 heterodimer (Jensen et al., 1998). Although it is difficult to decouple the additional density from the conformational changes in the polymer-
ase, a peak of positive density indicates that RPB4/ RPB7 is located between RPB5 and the base of the clamp (Cramer et al., 2000). The surface of the RNAPII 10 subunit core is highly negatively charged (Cramer et al., 2001). Aside from the active site cleft, which binds the DNA template and the DNA/RNA hybrid, the only surfaces that are either neutral or positively charged are the saddle and the base of the clamp. The highly asymmetric charge distribution of the E/F heterodimer (Figure 3) suggests a likely orientation for the complex (Figure 4), with the F subunit on the outside, maintaining the overall negative charge on the external surface of the RNAP core. Any other alternative would place highly negative surfaces (the polymerase core and the back of the E/F dimer) in close contacts. An additional argument supports the proposed orientation: in the yeast homolog a long insertion loop is located between helices H1 and H2, suggesting that this side of the complex is unlikely to be in contact with the core. RPB7 Is Positioned to Interact with the Nascent RNA Transcript The presence of an S1 motif in the sequence of the eukaryotic RPB7 and archaeal E subunit strongly suggests that the role of this subunit is to bind singlestranded nucleic acids. This has been shown in the case of the S. cerevisiae RPB4/RPB7 complex (Orlicky et al., 2001). Although a number of OB folds bind ssDNA as well as RNA, all the known members of the S1 subfamily bind to ssRNA, often in a sequence-nonspecific manner. In most of these proteins, RNA binding is spread over more than one domain, with the nucleic acid binding site forming a continuous surface. This is exemplified in the structure of NusA, which forms an extended RNA binding surface that spirals around the S1 motif and the KH domain (Worbs et al., 2001). If the truncated RNP fold in the N-terminal domain of subunit E does indeed bind nucleic acid, this subunit could contribute to a similar extended RNA binding surface. One argument in favor of this hypothesis is the fact that a partially conserved “path” can be identified along the surface of the molecule, leading from the S1 motif binding surface, through Lys 40, Asp 41, Arg 37, and Lys 3, down to the putative nucleic acid binding region close to the loop between strands A3 and A4. One interesting aspect of this path is the relatively high temperature factors displayed by the residues involved, suggesting that a certain degree of flexibility may be instrumental to nucleic acid binding. We favor a model in which the S1 motif of RPB7 interacts with the nascent RNA transcript, possibly assisted by the truncated RNP motif (Figure 4); this model is consistent with the predicted position of the heterodimer at the base of the clamp (Cramer et al., 2000), where the RNA exit groove is located. Apart from the emerging RNA transcript, the only other occurrence of single stranded nucleic acid in transcription is the noncoding DNA strand in the transcription bubble, which is likely to be almost entirely enclosed within the RNAP core. Moreover, this would require RPB4/RPB7 to be located in a position incompatible with the EM results. Furthermore, our model is in agreement with the observation that the RPB4/RPB7 heterodimer favors a
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Figure 4. Proposed Model for the Interaction of RPB4/RPB7 with the RNA Polymerase Core A schematic representation of the RNAPII 10 subunit core is shown (viewed in a similar orientation as the side view in Figures 3 and 6D of Cramer et al. (2000), with RPB5 shown in red and the mobile clamp, which closes onto the active side cleft upon nucleic acid binding, shown in green. The predicted exit path for the nascent RNA transcript is shown as a dashed line, and the proposed location of the heterodimer shown as a blue circle. The structure of the E/F complex is shown rotated by approximately 130⬚ around a vertical axis with respect to the orientation used in Figure 2A. The position and orientation of the heterodimer is consistent with a wide range of structural and biological results, including low-resolution studies on 2D crystals and the positioning of the S1 motif binding face onto the nascent RNA transcript. The negatively charged surface of the F subunit is positioned on the outside, the insertion in the yeast RPB4 structure can be easily accommodated, and RPB7 plays the main role in the interaction, in agreement with the biochemical and genetic results.
closed conformation of the polymerase around the DNA binding cleft and increases DNA binding (Jensen et al., 1998). Binding of RPB7 to the nascent transcript may further enhance the conformational changes triggered by the presence of the DNA/RNA hybrid, leading to a tightly bound and more stable transcribing complex. Indeed, a three-step model for transcription initiation has been proposed (Holstege et al., 1997), where the third step is a structural transition occurring after the synthesis of the first 10–11 ribonucleotides, accompanied by a switch from abortive to productive RNA synthesis and promoter clearance. Binding of the nascent transcript to RPB7 may be coupled to the movement of the clamp to help achieve this last conformational change leading to a stable transcribing complex. RPB4 Has a Stabilizing Role There have been a number of reports suggesting a specific role for the heterodimer, and particularly RPB4, in transcription during stress. However, no explicit correlation has ever been demonstrated between either RPB4 or RPB7 and the specific transcription of heat-shock or stress genes. Despite the normal growth rate, rpb4⌬ cells exhibit stress-unrelated transcriptional defects, showing that this subunit has a general role in efficient mRNA synthesis under normal growth; therefore the stress response anomalies may be secondary consequences of a general transcription defect and the consequent perturbation of cellular functions (Bourbonnais et al., 2000; Tan et al., 2000). It has also been shown that overexpression of RPB7 can partly suppress some of the rpb4⌬ phenotypes, and that this subunit can interact with the RNAPII in the absence of RPB4, albeit less efficiently, making RPB7 the critical component of the dimer (Sheffer et al., 1999). The role of RPB4 is likely to be to stabilize the conformation of RPB7. The structure of the E/F heterodimer
strongly argues in favor of this conclusion, since no particular feature seems to suggest a functional role for this subunit either in binding nucleic acid or interacting with the polymerase core. The F subunit packs against one side of E at the hinge between the N-terminal and C-terminal domains as a “protective belt.” RPB4 probably facilitates the recruitment of RPB7 to the polymerase core, by enhancing its stability and/or supplying additional contacts. Under stress, an efficient interaction between the polymerase core and RPB7 becomes crucial for cell viability and the stabilizing presence of RPB4 indispensable. Experimental Procedures Protein Expression and Purification Coexpression of a recombinant full-length M. jannaschii E/F complex was achieved using a bicistronic expression strategy in E. coli BL21 (DE3) cells using a pGEX-2TK vector (Pharmacia), with the F subunit produced as a fusion with glutathione S transferase (Werner et al., 2000). For crystallization experiments, the protein was equilibrated with crystallization buffer (20 mM MES [pH 6.5], 100 mM sodium chloride, and 10 mM -mercaptoethanol) and concentrated with a Centricon-10 micro-concentrator (Amicon) to a final protein concentration of 20–25 mg/ml. Crystallization and Data Collection Crystals were grown at 4⬚C by vapor diffusion in hanging drops, equilibrated against a well solution containing 1.3–1.6 M ammonium dihydrogen monophosphate and 100 mM Tris/HCl (pH 4.6). The diffraction pattern is consistent with the tetragonal space group P43 with cell dimensions of a ⫽ b ⫽ 92 A˚ and c ⫽ 91 A˚. A self-rotation function showed the presence of a noncrystallographic 2-fold axis relating two complexes in the asymmetric unit. All data were collected at 100 K at beamline ID14-4 of the European Synchrotron Radiation Facility (ESRF, Grenoble, France). The images were processed and integrated using MOSFLM and subsequently handled using the CCP4 package (Collaborative Computational Project Number 4, 1994). Data collection statistics are summarized in Table 1.
Structure of an Archaeal RPB4/RPB7 Complex 1143
Structure Solution and Refinement The structure was solved by the multiple isomorphous replacement method (Table 1), followed by solvent flattening and noncrystallographic symmetry averaging, and refined with the program CNS (Bru¨nger et al., 1998), using all reflections between 20 and 1.75 A˚. The final model has a crystallographic R-factor of 21.7% (Rfree⫽ 23.8% calculated using 5% of the data) with a bond length r.m.s. deviation from ideality of 0.006 A˚. The two complexes in the asymmetric unit are very similar and can be superposed with a root-mean-square deviation of 0.39 A˚ between equivalent ␣ carbons. Analysis of the model with PROCHECK (Laskowski et al., 1993) shows good geometry with all residues in the allowed regions of the Ramachandran plot. Acknowledgments This work was supported by a Wellcome Trust Grant to S.O, P.B., and R.O.J.W. We are very grateful to Gordon Leonard for assistance during data collection at ESRF. Figures 2, 3, and 4 were prepared using the programs MOLSCRIPT (Esnouf, 1997), RASTER3D (Merritt and Bacon, 1997), and GRASP (Nicholls et al., 1991).
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Received July 23, 2001; revised September 26, 2001.
McKune, K., Richards, K.L., Edwards, A.M., Young, R.A., and Woychik, N.A. (1993). RPB7, one of two dissociable subunits of yeast RNA polymerase II, is essential for cell viability. Yeast 9, 295–299.
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