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1 doi: /nature21393 SUPPLEMENTARY TEXT SpMED Immuno-purification and subunit localization Wild-type and subunit deletion mutant SpMEDs (Extended Data Table 1) were immunopurified through TAP-tagged or FLAG-tagged subunits, essentially as described for ScMED 1. We used SDS-PAGE (Extended Data Fig 1b) and MudPIT mass spectrometry to determine subunit composition, and EM to localize specific SpMED subunits. It is generally acknowledged that SpMED does not include Tail module subunits Med5 and Med16, but the presence of 4 other subunits had to be investigated. ORFs corresponding to possible Med2, Med9, and Med15 SpMED orthologs had been identified, as had been a possible ortholog of HsMED MED27. MudPIT mass spectrometry confirmed the presence of Med2, Med9, Med15, and Med27 in our SpMED preparations (Supplementary Information Table 1). EM analysis of ΔMed2 SpMED localized Med2 to partially ordered Tail density (Extended Data Fig 1c), in a position corresponding to the position of Med2 in ScMED 1. We were unable to obtain a ΔMed15 strain, but Med15 interacts directly with Med2 2 and diffuse density in 2D and 3D SpMED maps points to a poorly ordered Med2/Med15 Tail. Consistent with biochemical evidence pointing to MED27 interaction with several Head module subunits in human Mediator 1, and a Head subunit in direct contact with Med20 in SpMED 3, EM analysis of ΔMed27 SpMED particles localized some Med27 density to the distal end of Med20, connecting the Med18-Med20 Head jaw to Med17 (Extended Data Fig 1c). Further experiments will be needed to definitely establish the location of Med27 and determine whether an extension of Med27 to the Tail could form a weak connection between purported Med27 density in the Head and Tail modules apparent in SpMED class averages (Extended Data Fig 1d). SpMED cryo-em analysis SpMED cryo-em specimens prepared on perforated carbon supports were imaged on a Krios Titan 300kV microscope (FEI) using a K2 Summit (Gatan) direct electron detector (Extended Data Fig 1e). Through a combination of template-based particle picking 4, 2D image clustering, 3D image classification, and iterative alignment carried out with RELION 5, we found that free Mediator, free RNAPII, Mediator-RNAPII holoenzyme (multiple Mediator-RNAPII contacts), and Mediator with RNAPII loosely bound near the hook, were all present in the cryo-em specimens (Methods, Extended Data Fig 2a). Mediator, RNAPII, and holoenzyme particles were stable in conformation and suitable for high-resolution cryo-em analysis. The position of RNAPII loosely bound near the hook was highly variable. Structure of the Mediator-RNAPII holoenzyme Images of holoenzyme particles (multiple Mediator-RNAPII contacts) in cryo-em samples showed RNAPII in a single, well-defined orientation (Extended Data Fig 2b). Previous observation of variability in RNAPII orientation in images of 1

2 RESEARCH SUPPLEMENTARY INFORMATION holoenzyme particles in stained samples 6,7 must have resulted from limitations of preservation in stain, as mild crosslinking prior to staining results in holoenzyme particles that show only the Mediator-RNAPII interaction mode detected in cryo- EM samples. There were no appreciable differences between a 4.3 Å resolution cryo-em map of Sp RNAPII we calculated from images of free polymerase present in our Sp Mediator samples (not shown), the RNAPII portion of the Sp holoenzyme map, and the published X-ray structure of Sp RNAPII 8 (Extended Data Fig 5e-h). Therefore, the atomic models of RNAPII and individual Mediator modules were fitted into the holoenzyme map and the Mediator portion was refined to obtain a holoenzyme atomic model (Fig 4a and Extended Data Movie 2). Mediator-RNAPII contacts in the Sp holoenzyme, a Sc core MED-ITC complex, and a Sc MED-PIC complex The Mediator-RNAPII interactions in our 7 Å Sp holoenzyme structure generally involve the same Mediator and polymerase domains involved in the interactions observed in a ~10Å cryo-em structure of a recombinant S cerevisiae core Mediator subcomplex interacting with an initial transcribing complex including RNAPII and a minimal subset of basal factors (Sc core MED-ITC) 9, and in a lowresolution cryo-em map of a Mediator-preinitiation (MED-PIC) complex 10 (Extended Data Fig 5i-k). This overall correspondence, despite differences in the organism studied (S cerevisiae vs S pombe), and in complex composition and assembly (holoenzyme particles purified directly from S pombe cells, vs. a partial recombinant Mediator assembled in vitro with a partial set of purified basal factors and a promoter, or a full purified Mediator incubated with a pre-formed complete PIC), points to the importance of the conserved interactions that were observed. A critical contribution from our study is that the higher resolution of our holoenzyme and Mediator cryo-em maps allows for a detailed understanding of Mediator-RNAPII interactions and, from a mechanistic perspective, the Mediator rearrangements that make those interactions possible. We identified four Mediator-RNAPII contacts. First, polymerase subunits Rpb3/Rpb11 interact with Med20 in the Head module. Rpb3 s cysteine 92, mutated in a temperature-sensitive S cerevisiae strain showing decreased RNAPII interaction with Mediator 11, sits precisely at the interface with Med20 (Extended Data Fig 6b), consistent with reported involvement of Rpb3 11 in Mediator RNAPII interaction and a Med20 effect on transcription activity 12. Second, Rpb4 (residues ) in the Rpb4/7 stalk contacts a portion of Med17 near the N-terminus (residues ) (Extended Data Fig 5g-h). Rpb4/Rpb7 density is comparatively weak in the holoenzyme map, but increases after focused refinement, pointing to Rpb4/Rpb7 mobility, not dissociation. These Rpb3 and Rpb4 contacts are consistent with a paramount role for the Head in polymerase interaction 13. Third, density matching the position of a CTD peptide in the crystal structure of a ScMED Head-CTD peptide complex 14 is apparent in a noticeably narrower gap between the Middle s knob and the Head s neck domains (Extended Data Fig 6c and 6i). We reported localization of a GST- 2

3 RESEARCH labeled CTD to the corresponding portion of ScMED 6. Finally, the polymerase Rpb1 foot forms a close contact with the four-helix bundle formed by the N- termini of Med4 and Med9 in the Middle module (Extended Data Fig 6d). A detailed comparison with the structure of the MED-PIC complex was precluded by its low resolution. To compare in more detail the Sc core MED-ITC structure (EMDB-2786) to our holoenzyme structure, we started by segmenting EMDB into Mediator modules, RNAPII, GTFs, and nucleic acid (Extended Data Fig 6e). We then fitted the Mediator portion of our holoenzyme map (without changing the relative positions of the Sp MED modules) into the Mediator portion of EMDB-2786 and found that the holoenzyme conformation of Sp MED matches the holoenzyme conformation of Sc MED. There are some differences in Med14 and Middle conformation, but the relative position of the modules is the same (Extended Data Fig 6f). The conformation of the Head is especially similar. The conformation of RNAPII is also the same in our Sp holoenzyme and in EMDB-2786 (i.e., no change in RNAPII conformation upon interaction with Mediator on either system, or upon interaction with factors in EMDB-2786). However, the polymerase orientations are rotated with respect to one another (Extended Data Fig 6g). The Med20-Rpb3 contact (Interaction I) in Sp is essentially the same as in Sc (Extended Data Fig 6h) and seems to act as a pivot point for polymerase rotation, with differences in the position of corresponding portions of RNAPII in EMDB-2786 and SpHolo increasing with increasing distance from the Med20-Rpb3 contact. Interaction of the CTD between the Head and the Middle s knob (Interaction III) is the same in the X-ray structure of a Head-CTD complex 14, in EMDB-2786, and in our SpHolo structure. Our Sp holoenzyme structure shows additional density in a gap between the Head s neck and the Middle s knob closely matching the position of a Head-bound CTD peptide in the X-ray structure of a Head-CTD complex 14. (Extended Data Figs 6c and 6i). Relative rotation of RNAPII with respect to the Med20-Rpb3 contact explains observed differences in Rpb4-Head interaction (Interaction II), with Rpb4 contacting only Med17 in SpHolo, but contacting both Med17 and Med8 in EMDB-2786 (Extended Data g-s 5h and 6j). It also explains the considerable difference in Med9-Rpb1 interaction (Interaction IV), with a very tight contact in the Sp holoenzyme, but no contact in EMDB-2786 (Extended Data Figs 6d and 6k). The position and conformation of the Middle module are very similar in the Sc core MED-ITC structure and our Sp holoenzyme (Extended Data Fig 6f), but the absence of some Mediator subunits in the Sc core MED-ITC could explain differences between contacts in our holoenzyme structure and in the Sc core MED-ITC. For example, Med1, which interacts very extensively with Med4/9 and could influence their interaction with polymerase or stability of the Middle module position, was not included in their core Sc MED. A role for Med1 in promoting Mediator interaction with RNAPII would be consistent with reported correlation 3

4 RESEARCH SUPPLEMENTARY INFORMATION between Med1 and RNAPII interaction with Mediator 15. Alternatively, RNAPII rotation and breaking of the Rpb1-Med4/9 contact could result from the presence of TFIIB, which seems to make some contacts with Med18 in the core ScMED- ITC. Also, a clear Rpb1-Med4/9 contact is apparent in the cryo-em map of a ScMED-PIC complex 10 (Extended Data Fig 5k). Conservation of Mediator rearrangements in the holoenzyme and possible role of further Mediator rearrangements. Rearrangements in Med14 facilitate a considerable change in conformation between free S pombe Mediator and holoenzyme. We reported an analogous change in conformation between the free and holoenzyme forms of S cerevisiae Mediator 1 based on comparison of lower-resolution Mediator and holoenzyme structures. The close correspondence between the Mediator portion of the Sc core MED-ITC structure and the holoenzyme portion of our Sp holoenzyme map (Extended Data Fig 6f) already suggests changes in S cerevisiae Mediator conformation upon holoenzyme formation. To further investigate differences between the free and holoenzyme conformations of Sc Mediator we compared the Mediator portion of the core Sc core MED-ITC structure (EMDB-2786) to the low (~16-18Å) resolution cryo-em map of S cerevisiae Mediator 1. An alignment based on the Head modules clearly shows a change in Sc Mediator conformation upon holoenzyme formation (Extended Data Fig 7b), matching the one we had previously described 1, and the one we have now seen after comparing higher resolution free and holoenzyme forms of S pombe Mediator (Fig 4b). The same rearrangement in Mediator conformation is evident in the low-resolution MED- PIC map (Extended Data Fig 7i). Comparatively low density for the Tail module in the Sp Mediator and holoenzyme cryo-em maps results from high mobility of the Tail, which connects to the rest of Mediator through the third Med14 repeat (RM3). Secondary structure similarity between all three Med14 repeat motifs suggests that the third repeat (RM3) should be at least as flexible as RM1 and RM2 (Extended Data Fig 4a-b). Tail repositioning triggered by binding of a recombinant Gcn4 activator to Med15 in Sc MED 1 is likely facilitated by malleability of the RM3 Med14 C- terminal repeat. We found a fraction of Sp holoenzyme particles in which Tail repositioning can result in Tail-RNAPII contacts (not shown), and similar changes in Tail conformation could facilitate Tail interaction with regulatory elements bound to upstream DNA, further emphasizing Med14 s critical importance. Calculation of the PIC-TFIIK cryo-em map To obtain a map of the S cerevisiae PIC complex with TFIIK density we used the same image sorting procedures described for calculation of cryo-em maps of S pombe complexes. We started with the same PIC image dataset used for calculation of the published PIC map 16. Image sorting for the published PIC map was carried out using a mask derived from an initial low-resolution cryo-em map, which we assumed included all PIC density. We repeated all image sorting and classification without the use of any mask and increased the number of classes 4

5 RESEARCH (8 classes) used during 3D image classification using Relion 5. This allowed us to identify a subset of 1855 PIC images (corresponding to ~3% of the initial 64,623 images) that showed clear density matching the expected size and location (adjacent to Rad3) of TFIIK (Extended Data Fig 9b). After 3D refinement, the final PIC-TFIIK cryo-em had a resolution of 14.7 Å (Extended Data Fig 9d). 1 Tsai, K. L. et al. Subunit architecture and functional modular rearrangements of the transcriptional mediator complex. Cell 157, , (2014). 2 Beve, J. et al. The structural and functional role of Med5 in the yeast Mediator tail module. J Biol Chem 280, , (2005). 3 Linder, T. et al. Two conserved modules of Schizosaccharomyces pombe Mediator regulate distinct cellular pathways. Nucleic Acids Res 36, , (2008). 4 Lander, G. C. et al. Appion: an integrated, database-driven pipeline to facilitate EM image processing. J Struct Biol 166, , (2009). 5 Scheres, S. H. RELION: implementation of a Bayesian approach to cryo- EM structure determination. J Struct Biol 180, , (2012). 6 Tsai, K. L. et al. A conserved Mediator-CDK8 kinase module association regulates Mediator-RNA polymerase II interaction. Nat Struct Mol Biol 20, , (2013). 7 Davis, J. A., Takagi, Y., Kornberg, R. D. & Asturias, F. A. Structure of the yeast RNA polymerase II holoenzyme: Mediator conformation and polymerase interaction. Mol Cell 10, , (2002). 8 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, , (2009). 9 Plaschka, C. et al. Architecture of the RNA polymerase II-Mediator core initiation complex. Nature 518, , (2015). 10 Robinson, P. J. et al. Structure of a Complete Mediator-RNA Polymerase II Pre-Initiation Complex. Cell 166, e1416, (2016). 11 Soutourina, J., Wydau, S., Ambroise, Y., Boschiero, C. & Werner, M. Direct interaction of RNA polymerase II and mediator required for transcription in vivo. Science 331, , (2011). 12 Lariviere, L. et al. Structure-system correlation identifies a gene regulatory Mediator submodule. Genes Dev 22, , (2008). 13 Takagi, Y. et al. Head module control of mediator interactions. Mol Cell 23, , (2006). 14 Robinson, P. J., Bushnell, D. A., Trnka, M. J., Burlingame, A. L. & Kornberg, R. D. Structure of the Mediator Head module bound to the carboxy-terminal domain of RNA polymerase II. Proc Natl Acad Sci U S A 109, , (2012). 15 Zhang, X. et al. MED1/TRAP220 exists predominantly in a TRAP/ Mediator subpopulation enriched in RNA polymerase II and is required for ER-mediated transcription. Mol Cell 19, , (2005). 16 Murakami, K. et al. Structure of an RNA polymerase II preinitiation complex. Proc Natl Acad Sci U S A 112, , (2015). 5