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. 2010 Feb 17;29(4):717-26.
doi: 10.1038/emboj.2009.401. Epub 2010 Jan 21.

Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry

Zhuo Angel Chen  1 , Anass Jawhari, Lutz Fischer, Claudia Buchen, Salman Tahir, Tomislav Kamenski, Morten Rasmussen, Laurent Lariviere, Jimi-Carlo Bukowski-Wills, Michael Nilges, Patrick Cramer, Juri Rappsilber
Affiliations

Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry

Zhuo Angel Chen et al. EMBO J. .

Abstract

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.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
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.
Figure 2
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.
Figure 3
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.
Figure 4
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).
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.
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