Architecture of the RNA polymerase II–TFIIF complex revealed by cross-linking and mass spectrometry
2010; Springer Nature; Volume: 29; Issue: 4 Linguagem: Inglês
10.1038/emboj.2009.401
ISSN1460-2075
AutoresZhuo A. Chen, Anass Jawhari, Lutz Fischer, Claudia Buchen, Salman Tahir, Tomislav Kamenski, Morten Rasmussen, Laurent Larivière, Jimi Wills, Michaël Nilges, Patrick Cramer, Juri Rappsilber,
Tópico(s)Genomics and Phylogenetic Studies
ResumoArticle21 January 2010Open Access Architecture of the RNA polymerase II–TFIIF complex revealed by cross-linking and mass spectrometry Zhuo Angel Chen Zhuo Angel Chen Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, The University of Edinburgh, Edinburgh, UK Search for more papers by this author Anass Jawhari Anass Jawhari Department of Biochemistry, Gene Center and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Lutz Fischer Lutz Fischer Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, The University of Edinburgh, Edinburgh, UK Search for more papers by this author Claudia Buchen Claudia Buchen Department of Biochemistry, Gene Center and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Salman Tahir Salman Tahir Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, The University of Edinburgh, Edinburgh, UK Search for more papers by this author Tomislav Kamenski Tomislav Kamenski Department of Biochemistry, Gene Center and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Morten Rasmussen Morten Rasmussen Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, The University of Edinburgh, Edinburgh, UK Search for more papers by this author Laurent Lariviere Laurent Lariviere Department of Biochemistry, Gene Center and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Jimi-Carlo Bukowski-Wills Jimi-Carlo Bukowski-Wills Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, The University of Edinburgh, Edinburgh, UK Centre for Systems Biology, The University of Edinburgh, Edinburgh, UK Search for more papers by this author Michael Nilges Michael Nilges Department of Structural Biology and Chemistry, Institut Pasteur, Paris, Cedex, France Search for more papers by this author Patrick Cramer Patrick Cramer Department of Biochemistry, Gene Center and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Juri Rappsilber Corresponding Author Juri Rappsilber Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, The University of Edinburgh, Edinburgh, UK Search for more papers by this author Zhuo Angel Chen Zhuo Angel Chen Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, The University of Edinburgh, Edinburgh, UK Search for more papers by this author Anass Jawhari Anass Jawhari Department of Biochemistry, Gene Center and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Lutz Fischer Lutz Fischer Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, The University of Edinburgh, Edinburgh, UK Search for more papers by this author Claudia Buchen Claudia Buchen Department of Biochemistry, Gene Center and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Salman Tahir Salman Tahir Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, The University of Edinburgh, Edinburgh, UK Search for more papers by this author Tomislav Kamenski Tomislav Kamenski Department of Biochemistry, Gene Center and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Morten Rasmussen Morten Rasmussen Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, The University of Edinburgh, Edinburgh, UK Search for more papers by this author Laurent Lariviere Laurent Lariviere Department of Biochemistry, Gene Center and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Jimi-Carlo Bukowski-Wills Jimi-Carlo Bukowski-Wills Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, The University of Edinburgh, Edinburgh, UK Centre for Systems Biology, The University of Edinburgh, Edinburgh, UK Search for more papers by this author Michael Nilges Michael Nilges Department of Structural Biology and Chemistry, Institut Pasteur, Paris, Cedex, France Search for more papers by this author Patrick Cramer Patrick Cramer Department of Biochemistry, Gene Center and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Juri Rappsilber Corresponding Author Juri Rappsilber Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, The University of Edinburgh, Edinburgh, UK Search for more papers by this author Author Information Zhuo Angel Chen1, Anass Jawhari2, Lutz Fischer1, Claudia Buchen2, Salman Tahir1, Tomislav Kamenski2, Morten Rasmussen1, Laurent Lariviere2, Jimi-Carlo Bukowski-Wills1,3, Michael Nilges4, Patrick Cramer2 and Juri Rappsilber 1 1Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, The University of Edinburgh, Edinburgh, UK 2Department of Biochemistry, Gene Center and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Munich, Germany 3Centre for Systems Biology, The University of Edinburgh, Edinburgh, UK 4Department of Structural Biology and Chemistry, Institut Pasteur, Paris, Cedex, France *Corresponding author. Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, The University of Edinburgh, King's Bldgs, Edinburgh EH9 3JR, UK. Tel.: +44 131 651 7056; Fax: +44 131 650 5379; E-mail: [email protected] The EMBO Journal (2010)29:717-726https://doi.org/10.1038/emboj.2009.401 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Higher-order multi-protein complexes such as RNA polymerase II (Pol II) complexes with transcription initiation factors are often not amenable to X-ray structure determination. Here, we show that protein cross-linking coupled to mass spectrometry (MS) has now sufficiently advanced as a tool to extend the Pol II structure to a 15-subunit, 670 kDa complex of Pol II with the initiation factor TFIIF at peptide resolution. The N-terminal regions of TFIIF subunits Tfg1 and Tfg2 form a dimerization domain that binds the Pol II lobe on the Rpb2 side of the active centre cleft near downstream DNA. The C-terminal winged helix (WH) domains of Tfg1 and Tfg2 are mobile, but the Tfg2 WH domain can reside at the Pol II protrusion near the predicted path of upstream DNA in the initiation complex. The linkers between the dimerization domain and the WH domains in Tfg1 and Tfg2 are located to the jaws and protrusion, respectively. The results suggest how TFIIF suppresses non-specific DNA binding and how it helps to recruit promoter DNA and to set the transcription start site. This work establishes cross-linking/MS as an integrated structure analysis tool for large multi-protein complexes. Introduction Protein crystallography has been the primary source of structural insights into multi-protein complexes for decades. However, only homogenous, stoichiometric, stable, and rigid complexes that are available in sufficient amounts generally form crystals of sufficient quality for X-ray analysis. Therefore, core complexes are often resolved by crystallography whereas the position of additional, peripheral factors remains elusive. Cross-link analysis can provide positional information on flexible, transient, and modular higher-order multi-protein complexes, by mapping regions of spatial proximity. Cross-linking and mass spectrometry (MS) have first been used for the analysis of a multi-protein complex a decade ago (Rappsilber et al, 2000). After long development (Sinz, 2006) cross-link sites are now identified by database searches in a similar way to protein modification sites (Maiolica et al, 2007; Rinner et al, 2008). This revealed the organization of the 180 kDa Ndc80 complex, the largest complex analysed to date, at peptide resolution (Maiolica et al, 2007) and guided the X-ray analysis of the complex (Ciferri et al, 2008). Here, we show that cross-linking can be used to study even larger complexes in synergy with established structural biology techniques. We applied our approach to a major unresolved question in molecular biology, the structure of the RNA polymerase II (Pol II) transcription initiation complex. Transcription initiation at eukaryotic protein-coding genes requires Pol II and the basal transcription factors (TFs) IIB, -D, -E, -F, and -H (Reinberg et al, 1998). Although the crystal structure of the 12-subunit Pol II is known (Armache et al, 2005), structural information on the complex of Pol II with initiation factors remains limited (Kostrewa et al, 2009). Here, we investigate the structure of Pol II in complex with TFIIF. TFIIF was first identified based on its tight interaction with Pol II (Sopta et al, 1985). In the yeast Saccharomyces cerevisiae, about half of Pol II is bound by TFIIF (Rani et al, 2004). Yeast TFIIF comprises the essential subunits Tfg1 and Tfg2, and the non-essential subunit Tfg3 (Henry et al, 1994). Human TFIIF consists of homologues to Tfg1 (Rap74) and Tfg2 (Rap30), but lacks a Tfg3 homologue (Henry et al, 1994). Rap74 comprises an N-terminal region that binds Rap30 (Wang and Burton, 1995), a charged, central region, and a C-terminal domain that binds the phosphatase Fcp1(Chambers et al, 1995; Kobor et al, 2000). Rap30 comprises an N-terminal region that binds Rap74 (Yonaha et al, 1993), a central region that binds Pol II (Sopta et al, 1989; McCracken and Greenblatt, 1991), and a C-terminal domain (Garrett et al, 1992; Tan et al, 1994b). The N-terminal regions of Rap74 and Rap30 form a dimerization domain with a triple-barrel fold (Gaiser et al, 2000), whereas the C-terminal domains of Rap74 and Rap30 form winged helix (WH) domains (Groft et al, 1998; Kamada et al, 2001). TFIIF is required for initiation at TATA-containing and TATA-less promoters (Burton et al, 1988), reduces the affinity of Pol II to DNA (Garrett et al, 1992), and prevents interaction of Pol II with non-specific DNA (Killeen and Greenblatt, 1992). TFIIF is required for stable pre-initiation complex formation (Tan et al, 1995), and for normal start site selection (Pinto et al, 1994; Ghazy et al, 2004; Freire-Picos et al, 2005). In initially transcribing complexes, TFIIF stimulates phosphodiester bond formation and stabilizes a short DNA–RNA hybrid (Funk et al, 2002; Khaperskyy et al, 2008). During elongation in vitro, TFIIF reduces the time of Pol II pausing (Flores et al, 1989; Price et al, 1989; Bengal et al, 1991; Chafin et al, 1991; Tan et al, 1994b) and suppresses backtracking and RNA cleavage induced by TFIIS (Elmendorf et al, 2001; Zhang et al, 2003). To understand the multiple TFIIF functions, and the architecture of the Pol II initiation complex, detailed structural knowledge of the Pol II–TFIIF complex is required. Electron microscopy (EM) of complexes of Pol II with endogenous TFIIF and recombinant Tfg2 at 18 Å resolution suggested that Tfg2 extends along the polymerase cleft and Tfg1 binds around the Rpb4/7 subcomplex and the clamp on the Rpb1 side of the cleft (Chung et al, 2003). Site-specific radical-generating probing however placed TFIIF on the other side of the cleft near Rpb2 (Chen et al, 2007). Here, we used protein cross-linking coupled to MS to first analyse the free, 12-subunit Pol II, a complex of 513 kDa. Agreement of the data with the crystal structure shows for the first time that the method can be applied to such large complexes. This establishes cross-linking coupled to MS as a tool for the structural analysis of large multi-protein complexes. We then apply the approach to the Pol II–TFIIF complex that was purified as a stoichiometrically homogeneous complex from yeast cells using a new protocol. This complex comprises 15 polypeptides and has a total molecular weight of 670 kDa. The resulting detailed map of cross-links between Pol II and TFIIF, together with previous crystallographic data and molecular modelling, unravels the architecture of the Pol II–TFIIF complex and provides insights into the function of TFIIF during transcription. Results MS cross-link analysis of Pol II To test whether we could extend our cross-link analysis to large multi-protein complexes, we analysed the 12-subunit 513 kDa Pol II, for which a crystal structure is available (PDB 1WCM) (Armache et al, 2005). Pol II was obtained as described (Sydow et al, 2009). 30 μg of Pol II was subjected to cross-linking with the label-free cross-linker Bis (sulphosuccinimidyl) suberate (BS3, Thermo Fisher Scientific) (see Materials and methods). BS3 reacts with primary amines in lysine side chains and protein N-termini. The amines must be <11.4 Å apart, the maximal length of the BS3 spacer. Adding 16 Å to this, two times the length of a lysine side chain (6–6.5 Å) including an estimated coordinate error for mobile surface residues (1.5 Å), defines the maximal C-α distance of linkable lysine residues, 27.4 Å, when comparing our cross-link data with the available crystallographic data. We used a charge-based enrichment strategy for cross-linked peptides (Maiolica et al, 2007; Rinner et al, 2008) and high-resolution MS for peptide and fragment detection (see Materials and methods). We identified 146 linkage pairs in 429 mass spectra matching to cross-linked peptides (Supplementary Tables 1 and 2). In our subsequent analysis we focussed on those 106 linkage pairs that had both linked residues present in the Pol II structure. Our cross-link data reflected accurately the structural features of Pol II. The observed cross-links were significantly different from a random selection of all possible pairs in the structure (P-value of 3 × 10−87) (Figure 1D). The C-α distances of 99 pairs fell below 27.4 Å, 95 (90%) fell below 23 Å, 79 (75%) fell below 19 Å. Only five of our high-confidence links (see Materials and methods) and two of low confidence cannot be explained with the crystal structure. This is apparently because of cross-linking being conducted in solution, allowing internal movement of protein regions that are fixed at certain positions in the crystal structure because of crystal lattice restraints. Indeed, six of these seven links involve residues with high B-factors in or proximal to the mobile clamp domain of Pol II (Figure 1E; Supplementary Figure S2). Thus, only a single lower confidence link appears to be false. We conclude that our cross-link analysis returns accurate distance constrains in the context of a large, multi-protein complex, with an experimentally determined error rate in the order of 1%, with 1 of the 106 observed cross-links being false. Figure 1.MS-coupled cross-linking analysis of Pol II. (A) SDS–PAGE analysis and (B) native gel electrophoresis of Pol II and BS3 cross-linked Pol II. Cross-linked Pol II was excised from the SDS–PAGE gel and analysed (red box). A higher-order linkage product (asterisk) was excluded, most likely corresponding to a Pol II dimer, also observed on the native gel in the absence and increased in the presence of cross-linker (asterisk). (C) High-resolution fragmentation spectrum of a cross-linked peptide. The linkage site Rpb2 K228–Rpb2 K246 was observed in the cross-linked peptide SALEK(xl)GSR/K(xl)AAPSPISHVAEIR (m/z 615.8439, 4+). Extensive ion series for both peptides are observed in the high-resolution fragmentation spectrum and provide high confidence in the match. (D) C-α distance distribution for experimentally observed lys–lys pairs (red bars) and a random probability distribution (blue bars) within Pol II. The approximate cross-link limit for BS3 of 27.4 Å is indicated by a dashed line. Observed links falling below this limit are in agreement with the X-ray structure of Pol II (PDB 1WCM); observed links exceeding this limit are potentially in conflict with the known structure. (E) Zoom into 1WCM showing Rpb2 K228 and Rpb2 K246 (red sphere). The link spans 33.1 Å and is thus 5.7 Å longer than the maximal distance the cross-linker plus side chains of lysine can bridge (27.4 Å). The crystallographic B-factor is 128 Å2 for Rpb2 K228 and 180 Å2 for Rpb2 K246, indicating both residues as likely mobile. Both residues are in loop regions. Download figure Download PowerPoint Preparation and characterization of the Pol II–TFIIF complex To subject the even larger, scarce, and fragile Pol II–TFIIF complex to cross-linking, we established a new protocol for its large-scale preparation. Extensive attempts to obtain S. cerevisiae TFIIF after co-expression of its subunits in Escherichia coli were unsuccessful. Previous purification of endogenous Pol II–TFIIF complex resulted in low yields and partially degraded Tfg1 (Chung et al, 2003). We therefore prepared a yeast strain that over-expresses the three TFIIF subunits and contains a tandem affinity purification (TAP) tag on Tfg2 (see Materials and methods). We could obtain up to 2 mg of pure Pol II–TFIIF complex after TAP and size exclusion chromatography. Pol II–TFIIF complex preparations contained the 12 Pol II subunits and three TFIIF subunits in apparently stoichiometric amounts (Figure 2A). Pol II was not phosphorylated, as judged from western blotting with antibodies specific against phosphorylation at the C-terminal repeat domain (CTD) residues Ser2, Ser5, or Ser7 (Figure 2B). In an RNA extension assay (see Materials and methods and (Brueckner et al, 2007), the Pol II–TFIIF complex was as active as free Pol II (Figure 2C). Thus, the new protocol provided previously unavailable amounts of pure, homogeneous, and catalytically active yeast Pol II–TFIIF complex. Figure 2.Preparation and MS-coupled cross-linking analysis of the complete Pol II–TFIIF complex. (A) SDS–PAGE analysis of pure Pol II–TFIIF complex. Protein identity was confirmed by MS (not shown). (B) Western blot analysis of the phosphorylation state of the Pol II CTD residues Ser2, Ser5, and Ser7; 10 μl of 5- or 20-fold dilutions of pure Pol II and Pol II–TFIIF complexes at 1 mg/ml were subjected to 6% SDS–PAGE. After blotting on a nitrocellulose membrane (GE Healthcare), dual labelling was performed with antibodies that recognize unphosphorylated CTD (8WG16, green) and antibodies 3E10, 3EB, and 4E12 (Chapman et al, 2007), specific for phopshorylated CTD serines 2, 5, and 7 (Ser2P, Ser5P, and Ser7P, respectively). Yeast crude extract (CE) was used as control. (C) RNA extension assay of Pol II and Pol II–TFIIF in vitro (see Materials and methods). (D) SDS–PAGE analysis of Pol II–TFIIF complex and BS3 cross-linked Pol II–TFIIF complex. Cross-linked Pol II–TFIIF complex was excised from the SDS–PAGE gel in two bands and analysed (red box). (E) Native gel electrophoresis of BS3 cross-linked Pol II–TFIIF complex with BS3 cross-linked Pol II complex for comparison. The native gel shows absence of a dimer complex for BS3 cross-linked Pol II–TFIIF complex. (F) Cross-link map for TFIIF in complex with Pol II. Observed links from TFIIF to Pol II (dashed lines) are colour coded by the respective Pol II subunit. Links between TFIIF subunits (blue) and within TFIIF subunits (grey). For colour coding of domains in TFIIF see Figure 3A. Download figure Download PowerPoint Cross-link analysis of Pol II–TFIIF complex We next cross-linked and analysed the Pol II–TFIIF complex (Figure 2D–F), comprising 15 subunits with a total molecular weight of 670 kDa. Using 200 μg of purified complex allowed for elaborate fractionation and more comprehensive analysis. We identified by MS 402 linkage sites of which 220 fell within TFIIF and 182 between Pol II and TFIIF (Supplementary Tables 3 and 4). Data covering residue pairs within Pol II were again obtained but not included in the analysis. The quality of the MS data allowed confident assignment of 224 linkage sites and revealed a further 178 sites with lower confidence. There was no confidence bias between intra- and inter-protein links. Of the 220 linkage sites within TFIIF 149 were within proteins and 71 between proteins. In total, 253 inter-protein and 149 intra-protein links were identified. In comparison, the previous study on the Ndc80 complex had identified 13 inter-protein and 12 intra-protein links (Maiolica et al, 2007). This advancement in number of detected linkage pairs apparently results from improved MS equipment including high-resolution fragmentation spectra, an additional fractionation step, and the larger size of the analysed complex, providing more possible linkage sites. The cross-link data obtained here for Pol II and Pol II–TFIIF complex are the largest collection to date and will provide a valuable resource to understand method-specific aspects such as the fragmentation behaviour of cross-linked peptides. Yeast TFIIF domain structure To build a model of the Pol II–TFIIF complex, we first modelled domains of yeast TFIIF based on the three known domain structures for human TFIIF (Figure 3). We first obtained sequence alignments between the human and yeast sequences using HHPred (Soding et al, 2005) (Supplementary Figure S3). We then modelled the yeast domains with program MODELLER. The sequence conservation for the two WH domains was high, making modelling straightforward. The Tfg1 WH domain spans residues 673–728, whereas the Tfg2 WH domain spans residues 292–354 (Figure 3). For the Tfg1–Tfg2 dimerization domain, modelling was hampered because of low sequence conservation and uncertainty with respect to the N-terminal border of the domain in Tfg1 (Chen et al, 2007). The modelling suggested that the dimerization domain encompasses Tfg1 residues 98–400 with a non-conserved insertion at 167–305, and Tfg2 residues 55–227, with a non-conserved insertion at 144–192. Following the N-terminal dimerization domain, Tfg1 contains a ‘charged region’ (residues 400–510). Figure 3.TFIIF domain architecture. (A) Schematic representation of TFIIF subunits and domains. Links between TFIIF subunits (blue) and within TFIIF subunits (grey). (B, C, D) Cross-links confirm domain modelling of yeast sequences into the human crystal structures for (B) the Tfg1 WH domain, (C) the Tfg2 WH domain, and (D) the dimerization domain of Tfg1 (blue) and Tfg2 (red). Lysine residues (sphere for c-α atom) and observed links (dashed lines, red for high confidence, grey for low confidence, green for inter-protein Tfg1–Tfg2) with distance found in the respective homology model. Download figure Download PowerPoint The domain homology models, and the proposed domain structure of yeast TFIIF subunits, could be validated with the set of distance restraints within and between TFIIF subunits that were obtained as part of the cross-link analysis of the Pol II–TFIIF complex. The homology models for the three yeast TFIIF domains agree with the distance restraints provided by cross-links (Figure 3B–D). We observed eight cross-links within the dimerization domain, four within the Tfg1 WH domain, and 15 within the Tfg2 WH domain. In the domain models, the cross-linked residue pairs are all within the permitted distance of 27.5 Å for C-α atoms. Taken together, the cross-linking and modelling reveal that the two large yeast TFIIF subunits form three structured domains in the Pol II-bound state. The N-terminal regions of subunits Tfg1 and Tfg2 form a dimerization domain, whereas WH domains are present in the C-terminal regions of the subunits. Location of TFIIF on Pol II The cross-linking with the Pol II–TFIIF complex revealed an extensive network of proximities between TFIIF subunits Tfg1 and Tfg2 and the second largest Pol II subunit Rpb2, whereas only few cross-links were observed, which involve other subunits (Figure 2). TFIIF is positioned mostly on the Rpb2 side of the Pol II cleft (Figure 4; Supplementary Figure S4). The TFIIF dimerization domain is located at the Pol II lobe. The dimerization domain was placed manually on the Pol II surface, using only high-confidence cross-links and only cross-linking residues that were located either within the dimerization domain and Pol II structure or not more than 10 residues away in sequence. Enough spatial restraints were available to position and orient the dimerization domain (Figure 4A; Supplementary Figure S5 and Supplementary Table 5). One high-confidence cross-link is not satisfied by this position of the dimerization domain (Tfg1 394 to Rpb2 228). Satisfying this constraint moves the domain along its axis into the cleft, also agreeing with some previously reported evidence (Chen et al, 2007) (Supplementary Figure S6). In this position, sufficient space remains for the DNA to pass below the domain. Satisfying this constraint, however, conflicts a number of constraints at the other end of the domain. The data hence support two overlapping positions for the dimerization domain and indicate residual mobility for this domain. This could indicate an open and closed form for the cleft with the TFIIF dimerization domain acting as a lid, open to allow entry of the DNA and closed during initial transcription. Figure 4.Architecture of the Pol II–TFIIF complex. (A) The TFIIF dimerization domain has been positioned on the Pol II surface based on a series of cross-links between Pol II and the dimerization domain. Cross-link sites on the Pol II surface (slate and pink, matching the colour code of the dimerization domain), cross-link sites in TFIIF (sphere for C-α atom), cross-links used for positioning the dimerization domain (red dashed line) and for validation (green dashed line). For linkage sites that are absent from the Pol II structure or the model of the Tfg1–Tfg2 dimerization domain the nearest residue that is present is highlighted and labelled together with an asterisk (compare with Supplementary Table 3 and Supplementary Table 5). (B) Location of high-confidence cross-linking sites on Pol II surface coloured according to cross-linked TFIIF domains (represented in Figure 3). The dimerization domain has been placed on the Pol II surface; the location of other TFIIF regions is indicated. Two views are used, the top view and the side view, related by a 90° rotation around the horizontal axis. For linkage sites that are absent from the Pol II structure or the model of the Tfg1–Tfg2 dimerization domain the nearest residue that is present is highlighted (compare with Supplementary Table 3 and Supplementary Figures S4 and S5). Download figure Download PowerPoint TFIIF regions extending from the dimerization domain locate to neighbouring surfaces on Pol II. The Tfg1 region N-terminal to the dimerization domain cross-links to external 1 domain of Rpb2. The Tfg1 charged region C-terminal to the dimerization domain cross-links around the Rpb1 jaw at the downstream end of the cleft (Figure 5). Figure 5.Architecture of the Pol II initiation complex. Pol II is represented in top view. The path of the DNA in a closed promoter initiation complex is indicated as a thick grey line (Kostrewa et al, 2009). Pol II subunits (left) and domains (right) are highlighted in canonical colours. The position of TFIIF regions is indicated. The point of attachment of the linker to the CTD of Rpb1 is depicted as an arrow. Download figure Download PowerPoint The Tfg1 WH domain is apparently highly mobile, as no cross-links to Pol II were obtained. Our data on the Tfg2 WH domain and the dimerization domain show that cross-linking can capture dynamic structures. However, there is currently no data that establishes a limit beyond which interactions are too dynamic to be captured by cross-linking using N-hydroxysuccinimide esters, such as BS3 used in our study. The existence of an upper limit has been shown at least for formaldehyde cross-linking (Schmiedeberg et al, 2009). Our data within the Tfg1 WH domain show that we are principally able to cross-link this domain and detect such cross-links. Absence of data linking this domain to the rest of the complex indicates therefore that cross-linking requires a minimal amount of interaction that is not present in this case. The domain being held in close proximity to the complex through the Tfg1 linker region alone is insufficient for detectable cross-linking. The Tfg2 linker C-terminal to the dimerization domain extends along the Rpb2 protrusion over the side of Pol II. This path leads to cross-links of the Tfg2 WH domain with the protrusion on the upstream face of Pol II (Figure 5). In the free Pol II–TFIIF complex, the Tfg2 WH domain is apparently not restricted to this location, as cross-links to the Pol II wall and clamp were also obtained. These interactions on wall and clamp are likely dynamic as the same sites also cross-link to the Tfg2 linker and C-terminal region (Figure 4; Supplementary Figure S7) and emphasize the ability of cross-linking to capture such transient interactions. Additional density at this location was observed in the previous EM structure (Chung et al, 2003). However, this alternative location of the Tfg2 WH domain cannot be adopted in an initiation complex, as it overlaps with the path of the DNA (Chen and Hahn, 2004; Kostrewa et al, 2009).
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