doi:10.1038/nature11991 Supplementary Figure 1 - Refinement strategy for PIC intermediate assemblies by negative stain EM. The cryo-negative stain structure of free Pol II 1 (a) was used as initial reference after low-pass filtering to 60 Å (b) in a projection matching strategy for the generation of 3D reconstructions of negatively stained PIC samples: TBP-TFIIA-TFIIB-DNA-Pol II (c), as previous plus TFIIF (d), as previous plus TFIIE (e), as previous except in an open state (f). The negative stain TBP-TFIIA-TFIIB-DNA-Pol II-TFIIF-TFIIE model (e) after low-pass filtering to 60 Å (g) was used as initial reference for the generation of the 3D reconstruction of the PIC model containing TFIIH (h). Color code of the segmented densities corresponding to the different TFs is the same as in Fig. 1. WWW.NATURE.COM/NATURE 1
Supplementary Figure 2 - Cryo-EM data processing of the TBP-TFIIA-TFIIB-Pol II-DNA complex. (a) Representative raw micrograph. The top right corner shows the reference used for projection matching. (b) Representative reprojections of the final 3D model (top) matched to reference-free 2D class averages (bottom). (c) Euler angle distribution for the final refinement step. (d) Fourier Shell Correlation curve and estimated resolution using the 0.143 criteria. (e) Final cryo-em reconstruction shown in two different views with available atomic coordinates for proteins and a DNA model fitted to the density. The flexible portion of the DNA lacking corresponding EM density is shown in transparency. The color code for TFs and their corresponding segmented areas in the map is the same as for Fig. 1. (f) The color-coded local resolution estimation shows the range of resolutions within the cryo-em map. Within this particular PIC subcomplex, the only physical connection between Pol II and the rest of the complex is via TFIIB, whose N-terminal zinc ribbon domain fills the Pol II RNA exit channel. TFIIB s N-terminal cyclin fold, located near the wall domain of Pol II, interacts with the downstream BRE (BREd) element and with TBP, while its C-terminal domain binds the BREu element and TBP. The TFIIB linker could not be resolved within our cryo-em structure, probably due to its flexibility and/or the limited resolution of our reconstruction (12 Å). WWW.NATURE.COM/NATURE 2
Supplementary Figure 3 - Cryo-EM data processing of the TBP-TFIIA-TFIIB-Pol II-DNA- TFIIF complex. (a) Representative raw micrograph. The top right corner shows the reference used for projection matching. (b) Representative reprojections of the final 3D model (top) matched to reference-free 2D class averages (bottom). (c) Euler angle distribution for the final refinement step. (d) Fourier Shell Correlation curve and estimated resolution using the 0.143 criteria. (e) Final cryo-em reconstruction shown in two different views with available atomic coordinates for proteins and a DNA model fitted to the density. The color code for TFs and their corresponding segmented areas in the map is the same as for Fig. 1. (f) The color-coded local resolution estimation shows the range of resolutions within the cryo-em map. WWW.NATURE.COM/NATURE 3
Supplementary Figure 4 - Cryo-EM data processing of the TBP-TFIIA-TFIIB-Pol II-DNA- TFIIF-TFIIE complex. (a) Representative raw micrograph and reference used for projection matching. (b) Representative reprojections of the final 3D model (top) matched to reference-free 2D class averages (bottom). (c) Euler angle distribution for the final refinement step. (d) Fourier Shell Correlation curve and estimated resolution using the 0.143 criteria. (e) Final cryo-em reconstruction shown in two different views with available atomic coordinates for proteins and a DNA model fitted to the density. Three WH domains have been positioned relative to the Pol II clamp based on existing crosslinking data 2. (f) The color-coded local resolution estimation shows the range of resolutions within the cryo-em map. WWW.NATURE.COM/NATURE 4
Supplementary Figure 5 - Cryo-EM data processing of the TBP-TFIIA-TFIIB-Pol II-DNA- TFIIF-TFIIE complex in the open state. (a) Representative raw micrograph and reference used for projection matching. (b) Representative reprojections of the final 3D model (top) matched to reference-free 2D class averages (bottom). (c) Euler angle distribution for the final refinement step. (d) Fourier Shell Correlation curve and estimated resolution using the 0.143 criteria. (e) Final cryo-em reconstruction shown in two different views with available atomic coordinates for proteins and a DNA model fitted to the density. Three WH domains have been positioned relative the Pol II clamp based on existing crosslinking data 2. (f) The color-coded local resolution estimation shows the range of resolutions within the cryo-em map. WWW.NATURE.COM/NATURE 5
Supplementary Figure 6 Negative stain EM and data processing of the TBP-TFIIA-TFIIB- Pol II-DNA-TFIIF-TFIIE-TFIIH complex in the closed state. (a) Representative raw micrograph and reference used for projection matching. (b) Representative reprojections of the final 3D model (top) matched to reference-free 2D class averages (bottom). (c) Euler angle distribution for the final refinement step. (d) Fourier Shell Correlation curve and estimated resolution using the 0.143 criteria. (e) Final negative stain reconstruction shown in two different views with available atomic coordinates for proteins and a DNA model fitted to the density. Three WH domains have been positioned relative the Pol II clamp based on existing crosslinking data 2. Part of the density corresponding to TFIIH can be accommodated by the crystal of XPD and a homology model of XPB. (f) The color-coded local resolution estimation shows the range of resolutions within the negative stain map. WWW.NATURE.COM/NATURE 6
Supplementary Figure 7 Position of the TBP-TFIIA-TFIIB sub-complex relative to Pol II. A model of the TBP-TFIIB-Pol II subcomplex was proposed by superimposing the yeast crystal structure of Pol II/TFIIB with the crystal structure of TFIIB/TBP 3. We extended that model to include TFIIA, by superimposing TBP in the TBP-TFIIA crystal structure and this more complete model is shown in grey. The position of a model generated by optimizing the fit to the cryo-em densities of the Pol II-TBP-TFIIA-TFIIB complex using the three individual crystal structures is shown color coded as in the rest of the paper using light hues. The same, but using the cryo-em map for the Pol II-TBP-TFIIA-TFIIB-TFIIF complex is shown in brighter hues. Differences between the three models using Pol II as the common superimposable element are described by rotation angles and RMSDs. The position of the axis of rotation is indicated by the purple rod. Assembly of the TBP-TFIIB-TFIIA subcomplex within a PIC results in a rotation of individual proteins relative to TFIIB, as compared with the pair-wise crystal structures, which is further extended by the presence of TFIIF. The rotation axis of the TBP/TFIIA module is located between the two cyclin folds of TFIIB. Interestingly, mobility of the C-terminal cyclin fold of TFIIB has been shown to be involved in transcriptional activation 4,5. This structural difference results in a radially increasing root-mean-square deviation (RMSD) for the two models, especially for TFIIA. WWW.NATURE.COM/NATURE 7
Supplementary Figure 8 Comparison of the promoter DNA path with a previously proposed model. The DNA model fitted into our EM density (color coded as before) and a previously proposed model 3 (dark grey) are simultaneously shown by superimposing Pol II in the two models. Protein is color coded as in the rest of the paper but shown in transparency. WWW.NATURE.COM/NATURE 8
Supplementary Figure 9 Position of the RAP30/74 dimerization domain. The cryo-em density (mesh) for the dimerization domain is shown, out of the difference map obtained by subtracting the cryo-em structures of the PIC without TFIIF from either the map with TFIIF (a) or with both TFIIF and TFIIE (b). The RAP30/74 dimerization domain (PDB ID: 1F3U) and the Pol II domains nearby (PDB ID: 3K1F) are positioned based on rigid body docking into the density map (see also Fig. 2c and Supplementary Fig. 3e). According to our pseudo-atomic models, the RAP74 component of TFIIF is in contact with the RPB2 lobe, external 2, and RPB9 jaw, whereas RAP30 only interacts with the RPB2 external 2 domain. A oval indicates the clash between the RPB2 lobe and the RAP74 C-terminal α1 helix, a region shown to be important in both transcription initiation and elongation 6,7. In the crystal structure α1 was only fully resolved in one out of the four copies present 8. Intriguingly, the difference map between the densities with and without TFIIF shows extra density (dashed oval) that suggests a possible docking site for α1 upon interaction with Pol II (present both in (a) and (b)). In agreement with this idea, the adjacent charged C-terminal linker of the RAP74 dimerization domain has been shown to crosslink to the RPB1 jaw 9, and in the Pol I counterpart of the TFIIF dimerization domain, the A49/A34.5 dimerization module 10, the A49 C-terminal helix is flexible and pointing toward our proposed new C-terminal RAP74 position. WWW.NATURE.COM/NATURE 9
Supplementary Figure 10 Role of the Pol II protrusion domain in the stabilization of the PIC. Zoomed-in bottom view as in Fig. 2e showing the region around the protrusion domain of Pol II in the four cryo-em structures analyzed. The crystal structure for the protrusion domain of Pol II (PDB ID: 4BBR) is shown based on docking of the entire Pol II complex into cryo-em density maps of TBP-TFIIA-TFIIB-DNA-Pol II (a), as previous plus TFIIF (b), as previous plus TFIIE (c), as previous except for in the open state (d). Colors used in the segmentation of the map as in Fig. 1. TFIIF also contributes to the extra density seen linking the WH domain and the protrusion domain across the central cleft (see also Fig. 2d). We ascribe this density to the linker region of RAP30, immediately N-terminal to the WH domain, which had been shown to play an important but unknown role in both transcription initiation and elongation 11,12. A 10 Å scale bar is shown in (b) to depict the close proximity of the BREu and this linker region of RAP30, in agreement with previous crosslinking studies 13-15. WWW.NATURE.COM/NATURE 10
Supplementary Figure 11 Movement of the Pol II clamp domain during PIC assembly. The clamp domain of Pol II (yellow) needed to be docked as a separate piece into the different cryo-em densities obtained in this study: (a) closed TBP-TFIIA-TFIIB-DNA-Pol II; (b) as previous plus TFIIF; (c) as previous plus TFIIE; (d) as previous but in the open state. The clamp domain within the yeast crystal structure (PDB ID: 3K1F) is shown in dark grey to highlight the conformational rearrangement during PIC assembly and promoter opening. The rest of components of the PIC components are shown in light grey. WWW.NATURE.COM/NATURE 11
Supplementary Figure 12 - DNA binding interface on the closed conformation of Pol II. Left panel: EM density corresponding to promoter DNA in the TBP-TFIIA-TFIIB-Pol II-DNA- TFIIF-TFIIE complex structure is shown together with the PIC ribbon model as in Supplementary Figure 10. A 3-strand β sheet in the clamp head and 2-helix bundle at the tip of RPB5 jaw domain make direct contact with the promoter DNA flanking the INR element. Right panel: a cutaway view of the yeast Pol II crystal structure in surface representation. Surfaces with negative, neutral and positive electrostatic potentials are colored in red, white and blue, respectively. Regions of contact with the DNA, indicated by the dashed, black ovals, are positively charged. WWW.NATURE.COM/NATURE 12
Supplementary Figure 13 - Important structural elements at the promoter melting start site. The flexible TFIIB linker helix (PDB ID: 3K7A) and the TFIIF arm domain (PDB ID: 1F3U) are shown based on docking into the cryo-em density of the full crystal structures as rigid bodies. The positions of positively charged residues are colored in blue. The initial melting region of the promoter DNA, ~20bp downstream of TATA, is indicated. WWW.NATURE.COM/NATURE 13
Supplementary Figure 14 Structural similarity between the Pol II PIC and the Pol III system. Cryo-EM structures for the human Pol II PIC as described here and for yeast Pol III 16 are shown on the left and right, respectively. Highlighted are the common positions of elements in maroon and purple with respect to the polymerase core. There are a total of 17 proteins within Pol III and some of its specific subunits had been proposed to be counterparts of the Pol II general transcription factors 17. The C37/C53 dimer of Pol III would correspond to the dimerization domain of RAP74/30 in TFIIF, while C31/C82/C34 within Pol III are located in a similar position to that seen for the two subunits of TFIIE 16. WWW.NATURE.COM/NATURE 14
Supplementary Figure 15 Structural analysis of free human TFIIH. (a) The density for the TFIH core complex in the negative-stain reconstruction of the PIC was manually segmented (light pink) and used as the initial model for refinement of free TFIIH, after low-pass filtering to 60 Å. (b) Negative stain reconstruction of free TFIIH (magenta). The contours of the TFIIH core complex are superimposed on the corresponding views of the free TFIIH structure. The extra density is proposed to correspond to the CAK complex (c) Representative reprojections of the 3D model of the segmented TFIIH core, used as a reference (top), and of the free, full TFIIH (center), matched to reference-free 2D class averages of the free TFIIH (bottom). The more smeared aspect of the density corresponding to the CAK subcomplex reflects its flexible character. The yellows boxes correspond to equivalent views. WWW.NATURE.COM/NATURE 15
Supplementary Figure 16 The RPB1 CTD linker is in close proximity to the CAK subcomplex. The TFIIH containing PIC model is oriented as shown in the right panel of Fig. 5b. The S. cerevisiae Pol II structure within the PIC model has been replaced here by a recent S. pombe Pol II crystal structure, in which an segment of the CTD linker in RPB1 is visible 18. This additionally resolved linker is colored in dark red and the most C-terminal residue is shown as a sphere to indicate the position from where the rest of the CTD would extend. The CAK subcomplex from a separate negative-stain reconstruction of the apo-tfiih is shown in mesh (see Supplemental Fig. 15). The CTD would be positioned close to the proposed location of the CAK subcomplex based on our analysis. A homology model of the CDK7 (PDB ID: 1UA2)-Cyclin H (PDB ID: 1KXU) complex generated based on the CDK2-Cyclin A structure (PDB ID: 1FIN) was docked into the density assigned to the CAK. This density is linked to the core TFIIH structure by a continuous density that could be partially composed of the Mat1 coil-coiled domain, which has been shown to connect to both XPB and XPD 19. WWW.NATURE.COM/NATURE 16
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general transcription factors. Mol Biol Evol 27, 1035-1043, doi:10.1093/molbev/msp316 (2010). 18 Spahr, H., Calero, G., Bushnell, D. A. & Kornberg, R. D. Schizosacharomyces pombe RNA polymerase II at 3.6-A resolution. Proc Natl Acad Sci U S A 106, 9185-9190, doi:10.1073/pnas.0903361106 (2009). 19 Busso, D. et al. Distinct regions of MAT1 regulate cdk7 kinase and TFIIH transcription activities. J Biol Chem 275, 22815-22823, doi:10.1074/jbc.m002578200 (2000). WWW.NATURE.COM/NATURE 18