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1 doi: /nature10162 Architecture of the Mediator Head module Tsuyoshi Imasaki, Guillermo Calero, Gang Cai, Kuang-Lei Tsai, Kentaro Yamada, Francesco Cardelli, Hediye Erdjument-Bromage, Paul Tempst, Imre Berger, Guy Lorch Kornberg, Francisco J. Asturias, Roger D. Kornberg, and Yuichiro Takagi Supplementary Text 1: The initial crystals derived from the recombinant Head, expressed by the single virus co-infection method 10, diffracted poorly (YT, GC and RDK, unpublished), suggesting the necessity for further protein complex engineering. For this purpose, we implemented the MultiBac system 25 combined with the SLIC method 26 for efficient and robust protein complex engineering essential for crystallization trials 16. Limited proteolysis of the recombinant Head module identified a flexible region at the N-terminus of the Med17 subunit, and further a flexible loop region comprising residues in Med18 that was previously reported to impede crystallization of the Med18-Med20 complex 18. Supplementary Text 2: Crystals of the Head module diffracting up to 5 Å were obtained but there remained two major obstacles towards structure determination: First, the crystals were highly mosaic, and polymorphic with the longest unit cell axis varying by as much as 10 Å. Second, the crystals were almost perfectly merohedrally twinned. Optimization of crystallization conditions as well as post-crystallization treatment including dehydration, "" 1

2 RESEARCH resulted in isomorphous crystals with low mosaicity ( degrees) and a substantial reduction in the twinning rate (below 0.3) (Supplementary Tables 1 and 2). The SeMetlabeled crystals belong to space group P3221 and gave diffraction to 4.3 Å (Supplementary Table 1). The reduction of mosaicity significantly improved the signalto-noise (S/N) ratio of the selenium anomalous signal. This enabled us to perform SeMet SAD phasing, and, importantly, model building of virtually the entire Mediator Head module. Supplementary Text 3: Initial phases for the Mediator Head module were determined using two derivatives (i) Ta6Br14 by Single Isomorphous Replacement with Anomalous Scattering (SIRAS) and (ii) from K3Ir(NO3)6 using Single Anomalous Dispersion (SAD). The phases from each derivative were used to identify the SeMet anomalous difference Fourier peaks. Phases were further improved by manual model building followed by rigid-body refinement, inclusion of the SeMet sites, and recalculation of map coefficients using combined model and SeMet SAD data phases. After several rounds of this iterative process, the number of SeMet sites identified was consistent with 98 out of 141 possible methionines. To minimize bias from the model phases, final phases were re-calculated by SAD utilizing these 98 SeMet sites. This approach resulted in an excellent quality electron density map calculated to 4.3 Å resolution (Supplementary Fig. 2a,b). Three molecules of the Head module are present in the asymmetric unit of the crystal, and 98 (of 141 possible) selenium anomalous difference peaks higher than 3 $ were clearly detected 2 "#

3 RESEARCH (Supplementary Fig. 2b,c). Their distribution, especially within the known structure of the Med18-Med20 heterodimer where the Se sites completely corresponded to the known positions of methionine residues!#, compellingly validated the quality and utility of our electron density maps (Supplementary Fig. 2c). The distribution of the selenium anomalous peaks, as well as comparison with an available Med18-Med20 structure!# validated our approach and the quality of our electron density map. Supplementary Text 4: A series of N- and C-terminal Med17 deletions were introduced into a 10xHis-tagged version of Med17, followed by co-expression with the other six Head module subunits. In addition, internal Med17 deletion mutations were generated and tested to assess their effect on the stability of the resulting Head module assemblies (Supplementary Figs. 8-11). Strikingly, Med17 residues serve as a scaffold for an assembly region (Supplementary Figs. 9a, 10) in which altogether five subunits are clustered. The organization of this region was further investigated by characterization of internal deletion mutants. As expected, deletion of residues had no effect, as these residues fall within the dispensable N-terminal region (Supplementary Fig. 9a, lane 15). Deletion of residues resulted in the loss of subunits Med6-Med8-Med18-Med20 and in the formation of a minimal Head complex (Supplementary Fig. 9a, lane 16), whereas deletion of residues had no effect on complex assembly (Supplementary Fig. 9a, lane 17). These results are fully consistent with our crystal "$ 3

4 RESEARCH structure: A majority of the helix bundle encompasses Med17 residues and a linker encompasses Med17 residues which is dispensable for complex assembly. The 200 N-terminal (NTD), and 287 C-terminal residues (CTD) of Med17 appear to be largely dispensable for Head assembly, because deletion of these regions had no marked effect on Head subunit composition (Supplementary Fig. 9a, lanes 6 and 10, Supplementary Figs. 10,11) We pursued EM imaging to further characterize the NTD and CTD of Med17 and their roles in Head module structure. In agreement with previous sequence analysis that predicted intrinsic disorder for the Med17 NTD 43, little or no structural difference was found between EM projection structures of wild type Head module and the Med17 NTD deletion mutant (data not shown). As indicated by EM studies, deletion of the CTD led to a loss of the density corresponding to the fixed jaw (Supplementary Fig. 9b), clearly suggesting that the Med17 CTD is the main component of the Fixed jaw domain, consistent with our crystal structure. Supplementary Text 5: The Med11(#1-16)/Med18(#71-156) double mutant formed an incomplete Head which lacked the Med18-Med20 subcomplex (Supplementary Fig. 14, lane 5), whereas the same Med18(#71-156) mutant alone when expressed in the context of an intact Med11 subunit retains the ability to form the Med18-Med20 subcomplex (Supplementary Fig. 14, lane 3). Interestingly, a shorter deletion mutant of Med18 (# ), which contains the Med17 interacting region, in the context of the Med "%

5 RESEARCH (#1-16) mutant resulted in a stable and otherwise complete Head module (Supplementary Fig. 14, lane 4). Supplementary Text 6: Prior biochemical characterization has suggested that the Med6 subunit binds directly to the Med7-Med21 subunits in the Middle module of Mediator 44. Genetic studies suggested that the transcriptional activation signals targeted to several subunits of the Middle module appear to converge on the Med6 subunit 45. Docking of our Head module crystal structure into the cryo-em structure of the entire Mediator complex indicates that the Med6 subunit can connect to this portion of the Middle module (Supplementary Fig. 15). Taken together, this analysis suggests that the Med6 subunit may function as a critical interface between the Mediator Head and Middle modules, and transduce a mechanical signal from the Tail/Middle module to the Head module and onto Pol II. This model is further supported by a study of Med6 temperature sensitive mutations, where a total of six mutations disrupting every segment of the NTD of Med6 as well as the Med6 BD (Figure 1a, Supplementary Fig. 16b) resulted in a loss of Gal4 activated transcription in vivo 46. Supplementary Text 7:! Mediator was shown to stimulate the CTD kinase activity of TFIIH!'. To validate our model of the Head the Pol II CTD TFIIH interaction, we tested if the Head module alone is capable of stimulating CTD phosphorylation. Complete Mediator, purified from "& 5

6 RESEARCH source and used here as a control, stimulated phosphorylation of the CTD 3-6 fold (Supplementary Fig. 18 lanes 2-3). Next we repeated the experiment with our recombinant Head module. The Head module alone was capable of stimulating already 2-3 fold, clearly indicating the functional connection between the CTD, Head and TFIIH. In conclusion, Mediator Head stimulates phosphorylation, albeit not at the level of complete Mediator. This discrepancy likely attributes to the absence of the Middle module, which forms extensive contacts to Pol II #(', and thus stabilizes the Pol II- Mediator interaction. 6

7 RESEARCH Supplementary)Table 1 Data collection, phasing and refinement statistics for structure SeMet K3Ir(NO3)6 Ta6Br14 NatIz1-4 Data collection Space group P3221 P3221 P3221 P3221 Cell dimensions a = b (Å) c (Å) Wavelength Resolution (Å) No. unique reflections 71,174 32,691 17,188 72,566 Rmerge (%) 10.0(65.0) 12.7(78.6) 16.3 (61.0) 9.5 (54.5) I/$(I) 14.3(2.0) 19.8(1.8) 13.9 (1.3) 15.7 (2.2) Completeness (%) 98.6(97.8) 99.7(99.7) 99.8 (99.9) 97.7 (96.5) Redundancy 6.5(5.1) 13.4(10.3) 8.8(8.6) 6.5 (4.6) Resolution (Å) No. reflections 89,531 Rmerge (%) 8.4(58.0) I/$(I) 8.9(1.5) Completeness (%) 92.6(86.0) Refinement SeMet data Resolution (Å) No. reflections 82,491 Rwork/ Rfree (%) 34.5/37.3 No. atoms Protein 17,847 Selenium 98 B-factors Protein 130 R.m.s deviations Bond lengths (Å) Bond angles (º) 1.54 Values in parentheses are for the highest-resolution shell "( 7

8 RESEARCH Supplementary Table 2 Analysis of crystal twinning SeMet K3Ir(NO3)6 Ta6Br14 NatIz1-4 Twinning analysis Britton alpha H alpha ML alpha

9 RESEARCH Supplementary Table 3 Plasmids for complex expressions used in this study Name Description Source pyt49 pfl-10xhis-med17 (wt) 16 pyt52 pfl-med6-med8 16 pyt75 pspl-med20-med18 16 pyt110 pucdm-med6-med8 16 pyt111 pucdm-med22-med11 16 pyt114 pspl-med20-med18 (#71-156) This study pyt115 pspl-med20-med18 (# ) This study pyt120 pucdm-med6-med22-med11-med8 16 pyt147 pucdm-med22-med11 (#1-16) This study pyt151 pucdm-med6-med22-med18-med20-med11-med8 16 pyt165 pfl-10xhis-med17 (1-108) This study pyt166 pfl-10xhis-med17 (1-200) This study pyt167 pfl-10xhis-med17 (1-300) This study pyt168 pfl-10xhis-med17 (1-400) This study pyt169 pfl-10xhis-med17 (1-500) This study pyt170 pfl-10xhis-med17 (1-600) This study pyt171 pfl-10xhis-med17 ( ) This study pyt172 pfl-10xhis-med17 ( ) This study pyt173 pfl-10xhis-med17 ( ) This study pyt174 pfl-10xhis-med17 ( ) This study pyt289 pfl-10xhis-med17 (# ) This study pyt290 pfl-10xhis-med17 (# ) This study pyt291 pfl-10xhis-med17 (# ) This study pyt311 pfl-med22-med11 (15, 16, GS) This study pyt518 pspl-med6-med20-med18 (# )-Med8 This study #* 9

10 RESEARCH Supplementary Table 4 S. cerevisiae plasmids used in this study Name Description Source pyt264 Med17 (wt) 16 pyt271 Med17 ( ) This study pyt272 Med17 ( ) This study pyt273 Med17 ( ) This study pyt274 Med17 ( ) This study pyt275 Med17 ( ) This study pyt276 Med17 ( ) This study pyt265 Med17 (1-108) This study pyt266 Med17 (1-200) This study pyt267 Med17 (1-300) This study pyt268 Med17 (1-400) This study pyt269 Med17 (1-500) This study pyt270 Med17 (1-600) This study pyt282 Med17 (% ) This study pyt283 Med17 (% ) This study pyt284 Med17 (% ) This study pyt285 Med17 (% ) This study pyt286 Med17 (% ) This study 10

11 RESEARCH 1! 2! 3! 10xHis Med17Δ1-108! Med17Δ1-184! Med6C-6His! Med6! Med18Δ ! Med8! Med20! Med ! Med11! Med22! Supplementary Figure 1 Recombinant Head module complex is proteolytically trimmed during crystallization. Lane 1 shows ~6 µg of a crystallization sample of freshly prepared modified Head module containing Med17 (%1-108) and Med18 (% ), analyzed by 4-12% NuPAGE followed by staining with Coomassie Brilliant Blue. Note that recombinant Med6C-6His protein was supplemented to the complex. Lane 2 shows the contents of a crystal loaded on the same gel. A Head module crystal was fished from a crystallization drop, washed by transferring four times into fresh a 5 µl drops containing reservoir solution, and dissolved in denaturing gel loading sample buffer for SDS-PAGE analysis. Lane 3 shows the final drop used for washing the crystal as a control. Mass spectroscopic analysis evidenced that the N-terminal 76 residues of Med17 and the C- terminal 80 residues of Med6 were proteolyzed in the crystal. #" 11

12 RESEARCH a! b! c! M81! M80! M224! M78! M276! M182! M296! M204! M57! M117! Supplementary Figure 2 Electron density map and Selenium anomalous peaks on the Mediator Head module. (a) Map by Se SAD phasing. Maps are contoured at 1.5 $ and overlaid on the C! trace. (b) The anomalous positions of the Head module are superimposed onto the surface representation of the Head module structure model. Anomalous maps are contoured at 3.0 $ and colored in red. (c) Comparison between selenium anomalous peaks on the movable jaw domain, and the known positions of methionine residues in the Med18-Med20 structure ##

13 RESEARCH MNVTPLDELQWKSPEWIQVFGLRTENVLDYFAESPFFDKTSNNQVIKMQRQFSQLNDPNAAVNMTQNIMTLPDGKNGNLE Med6 EEFAYVDPARRQILFKYPMYMQLEEELMKLDGTEYVLSSVREPDFWVIRKQRRTNNSGVGSAKGPEIIPLQDYYIIGANI BH1 YQSPTIFKIVQSRLMSTSYHLNSTLESLYDLIEFQPSQGVHYKVPTDTSTTATAATNGNNAGGGSNKSSVRPTGGANMAT VPSTTNVNMTVNTMGTGGQTIDNGTGRTGNGNMGITTEMLDKLMVTSIRSTPNYI BH1 BH2 Med8 MSQSTASLVPEGNQGSLQEDVSFDFNGVPGQALDAVRMRLAQLTHSLRRIRDEMSKAELPQWYTLQSQLNVTLSQLVSVT BH3 40 BH BH5 80 STLQHFQETLDSTVVYPLPKFPTTSHESLVTTLLRKKNIPEVDEWMKYVRETSGVTTALLKDEEIEKLLQQDREITNWAR CTH TTFRNEYGKHDFKNEESLSEEHASLLVRDSKPSKPFNVDDVLKFTFTGEKPIITGSTSTSSSN BH1 BH2 Med11 MQVLNTKSETKQENETMQPPYIQERLKSLNDIETQLCSMLQEASQVTFIFGELKRGNESVKPQFENHVKQFYERLDKSTT CTH QLRKEIQLLDENVGTRLLPINVNKKALGQDTEKMEEQLDLLSAILDPSKSK MTTEDPDSNHLSSETGIKLALDPNLITLALSSNPNSSLHSPTSDEPVPESAGKADTSIRLEGDELENKTKKDNDKNLKFL KNKDSLVSNPHEIYGSMPLEQLIPIILRQRGPGFKFVDLNEKELQNEIKQLGSDSSDGHNSEKKDTDGADENVQIGEDFM BH1 EVDYEDKDNPVDSRNETDHKTNENGETDDNIETVMTQEQFVKRRRDMLEHINLAMNESSLALEFVSLLLSSVKESTGMSS BH2 Med17 MSPFLRKVVKPSSLNSDKIPYVAPTKKEYIELDILNKGWKLQSLNESKDLLRASFNKLSSILQNEHDYWNKIMQSISNKD Linker VIFKIRDRTSGQKLLAIKYGYEDSGSTYKHDRGIANIRNNIESQNLDLIPHSSSVFKGTDFVHSVKKFLRVRIFTKIESE DDYILSGESVMDRDSESEEAETKDIRKQIQLLKKIIFEKELMYQIKKECALLISYGVSIENENKVIIELPNEKFEIELLS LDDDSIVNHEQDLPKINDKRANLMLVMLRLLLVVIFKKTLRSRISSPHGLINLNVDDDILIIRPILGKVRFANYKLLLKK IIKDYVLDIVPGSSITETEVEREQPQENKNIDDENITKLNKEIRAFDKLLNIPRRELKINLPLTEHKSPNLSLMLESPNY CTH CNALIHIKFSAGTEANAVSFDTTFSDFKEVEDFLHFIVAEYIQQKKV #$ 13

14 RESEARCH MVQQLSLFGSIGDDGYDLLISTLTTISGNPPLLYNSLCTVWKPNPSYDVENVNSRNQLVEPNRIKLSKEVPFSYLIDETM Med18 MDKPLNFRILKSFTNDKIPLNYAMTRNILHNTVPQVTNFNSTNEDQNNSKHTEDTVNESRNSDDIIDVDMDASPAPSNES CSPWSLQISDIPAAGNNRSVSMQTIAETIILSSAGKNSSVSSLMNGLGYVFEFQYLTIGVKFFMKHGLILELQKIWQIEE AGNSQITSGGFLLKAYINVSRGTDIDRINYTETALMNLKKELQGYIELSVPDRQSMDSRVAHGNILI MGKSAVIFVERATPATLTELKDALSNSILSVRDPWSIDFRTYRCSIKNLPADVSKLMYSITFHHHGRQTVLIKDNSAMVT Med20 TAAAADIPPALVFNGSSTGVPESIDTILSSKLSNIWMQRQLIKGDAGETLILDGLTVRLVNLFSSTGFKGLLIELQADEA GEFETKIAGIEGHLAEIRAKEYKTSSDSLGPDTSNEICDLAYQYVRALEL BH1 BH2 Med22 MSNQALYEKLEQTRTILSVKLAELINMTTIADRNDDDEGSFAQENSELAVATTSVMMVNNQTMQLIKNVQDLLILTRSIK Linker CTH EKWLLNQIPVTEHSKVTRFDEKQIEELLDNCIETFVAEKTT Supplementary Figure 3 Secondary structure of the Mediator Head module subunits. The secondary structure elements of the Head Module subunits are depicted above the primary sequences. Helices are shown as cylinders, "-strands are shown as arrows, and loops are drawn as lines. The disordered regions are shown as dotted lines. Wide gray lines indicate regions where electron density was observed but could not be assigned reliably. #% 14

15 RESEARCH Fixed jaw domain N N 29 Med17 BH Med8 BH1 55 Joint N Med6 NTD Med8 BH4 Med8 BH Med8 BH Med6 BH Med22 BH Med22 BH C Med11 BH Med11 BH Med17 BH2 318 N Med11 NTD Med22 CTH C Med11 CTH C Med17 CTH N Med18 Loop 137 Med8 BH5 Bundle 169 Med8 CTH C Med18 Med20 Neck domain Movable jaw domain Supplementary Figure 4 Topology diagram of the Head module crystal structure. Helices are shown as cylinders, "-sheets as squares, linker regions are drawn as lines, disordered region are drawn as dotted lines. Joint is shown as a gray box labelled Joint. The N- and C- termini of each subunit are indicated. SeMet positions are shown as circles filled in purple. #& 15

16 RESEARCH Neck! Joint! Fixed jaw! Movable! Jaw! Supplementary Figure 5 Overall structure of the Head module of Mediator. A ribbon model of the Mediator Head module is seen from different angles. 16

17 RESEARCH a! b! Neck! Fixed jaw! Movable! Jaw! c! d! Supplementary Figure 6 Comparison of the X-ray structure with EM maps in different conformations. (a) The X-ray structure of the Mediator Head module is shown. (b-d) 3D reconstructions of the Head module in three different conformations characterized by single particle EM are depicted,, which are denominated "Collapsed" (b) "Closed" (c) and "Open" (d), respectively 16. #( 17

18 RESEARCH Supplementary Figure 7 Comparison between the X-ray and EM maps of the Head module. The X-ray structure is docked into the EM map of the Head in its closed conformation 16. The X-ray structure is shown as a ribbon diagram and also as a 15 Å resolution molecular map (both in blue), fitted inside the EM map of the Head (in gray mesh)

19 RESEARCH Med17! Med17 (1-108)! Med17 (1-200)! Med17 (1-300)! Med17 (1-400)! Med17 (1-500)! Med17 (1-600)! Med17 ( )! Med17 ( )! Med17 ( )! Med17 ( )! Med17 ( )! Med17 ( )! Med17 ( )! 1! 100! 200! 300! 400! 500! 600! 687! Complex! Assembly! +! +! +! +! +! +! +/ +! +/ +! Supplementary Figure 8 Schematic diagram of the N-, and C-terminal and internal deletion mutants of Med17 subunits and their effects on the Head module complex. Assemblies of the Head module Med17 mutants are illustrated. The results of the complex assembly are shown on the right column. (+): complex assembled; (+/-): partially assembled; (-): no assembly. $* 19

20 RESEARCH a! Med17! Core! Full! 1-108! 1-200! 1-300! 1-400! 1-500! 1-600! ! ! ! ! *! *! *! Core! Full! Δ ! Δ ! Δ ! *! Med18! Med6! Med8! Med20! Med11! Med22! 1! 2! 3! 4! 5! 6! 7! 8! 9! 10! 11!12! *! 13! 14! 15! 16! 17! b! WT! Med17 Δ ! Supplementary Figure 9 Biochemical and EM imaging analysis of Med17 subunit of the Head module (a) SDS-PAGE of recombinant Head module and mutant forms containing deletions in the Med17 subunit. Approx.6 µg of a peak fraction eluted from a Ni column were analyzed by 4-12% NuPAGE, followed by staining with Coomassie Brilliant Blue. Core (lanes 1, 13), and full Head modules (lanes 2,14); the C-terminal domain (CTD) deletion mutants (lanes 3-8); the N-terminal deletion mutants (lanes 9-12); the internal deletion mutants (lanes 15-17) are shown. The positions of full-length subunits in the gel are marked on the left. An asterisk indicates contaminants from insect cells that were copurified. (b) EM imaging analysis of the wild type Head module and mutant form containing the Med17 CTD deletion.class averages obtained after alignment of wild-type Head alone (left panel) and mutant Head with CTD deletion (middle and right panel) evidences the absence of density in the region corresponding to the Fixed jaw of the Head module $!

21 RESEARCH 1! 100! 200! 300! 400! 500! 600! 687! Med17! Med8! Med20! Med17 (1-400)! Med6 Med18! Med11! Med22! Med8! Med20! Med17 ( )! Med6 Med18! Med11! Med22! Med17 ( )! Med11! Med22! Med17 ( )! Med11! Med22! Med8! Med20! Med17 ( )! Med6 Med18! Med11! Med22! Supplementary Figure 10 Schematic illustration of subunit assembly of the Head module with different Med17 deletion mutants. Topographical diagram illustrating how the subunits of the Head module are assembled with the representative Med17 mutants, Med17 (1-400), Med17 ( ), Med17 ( ), Med17 (% ), Med17 (% ), are shown. $" 21

22 RESEARCH Fixed jaw! Neck! Movable! Jaw! Med17 ( )! core! mini! Med17 ( )! Med17 ( )! Med17 ( )! Med17 (1-400)! Supplementary Figure 11 Models of the subunit assembly of the Head module and mutant Head modules with containing different Med17 deletions mutants. Models illustrating how the subunits of the Head module and mutant Head modules are assembled. Respective Med17 mutants are Med17 (1-400), Med17 ( ), Med17 ( ), Med17 (% ), Med17 (% ), as indicated below the images. Models were generated from our crystal structure of the Mediator of Head module. $# 22

23 RESEARCH WT! ΔMed ! Transcripts/Template (x10 3 )! 1! 2! 3! 4! 5! 6! 7! Supplementary Figure 12 Transcription activity of wild-type Head module and the Med17 CTD deletion mutant. Transcription experiments were performed with srb4ts mutant extract at 30 o C as described 10 with addition of wild-type Head module (0, 0.5, 1.0, and 2.0 pmol in lanes 1-4), or of Med17 CTD deletion mutant (0.5, 1.0, and 2.0 pmol in lanes 5-7). Transcripts (~360 bp) separated by 6% denaturing PAGE and revealed by a fluorescent image analyzer (top) were quantified by fluorescent image analysis (bottom). WT: wild-type Head module, #CTD: C-terminal domain (CTD) deletion mutant of Med17-Head. $$ 23

24 RESEARCH Med17! Med17 (1-108)! Med17 (1-200)! Med17 (1-300)! Med17 (1-400)! Med17 (1-500)! Med17 (1-600)! 1! 100! 200! 300! 400! 500! 600! 687! Viability! +! Med17 ( )! Med17 ( )! Med17 ( )! Med17 ( )! Med17 ( )! Med17 ( )! Med17 ( )! Med17 ( )! Med17 ( )! Med17 ( )! Med17 ( )! +! +! +! Supplementary Figure 13 Schematic diagram of phenotypic analysis of Med17 deletion mutations in S. cerevisiae. Yeast shuttle vectors bearing Med17 deletion mutations illustrated were transformed into yeast strain Z572 by plasmid shuffling as described 15. The wild-type (WT) and mutant strains were grown in SC-Leu medium, spotted onto SC-Ura-Leu and SC +5-FOA plates and incubated at 30 C for overnight to assess viability. (+): viable; (-): inviable $% 24

25 RESEARCH Med18 Δ ! Med11 Δ1-16! Med18 Δ71-156! Core! Full! Med18 Δ71-156! Med11 Δ1-16! Med17! Med17! Δ1-108! Med18! Med6! Med8! Med20! Med11! Med22! 1! 2! 3! 4! 5! Med18! Δ ! Med18! Δ71-156! Supplementary Figure 14 SDS-PAGE analysis of Head module expression experiments with a combination of single and double deletion mutants of Med18 and Med11 in a Med17 (%1-108) deletion background. ~6 µg of a peak fraction from Ni affinity chromatography was analyzed by 4-12% NuPAGE, followed by staining with Coomassie Brilliant Blue. Shown are: Core module (lane 1), full module (lane 2), full module with Med18 (%71-156) mutant (lane 3), full module with Med11 (%1-16) and Med18 (% ) double mutants (lane 4), and full module with Med11 (%1-16) and Med18 (%71-156) double mutants (lane 5). $& 25

26 RESEARCH free! Mediator! Med6! Head Module! Supplementary Figure 15 Docking of our X-ray crystal structure into the Head portion of the Mediator cryo-em map. The fitting of the X-ray structure within the Head portion of the Mediator EM map is supported by the observation that the N-terminal portion of Med6 extending away from the neck can be uniquely positioned into a protrusion extending from the back of the Head in the cryo-em map. The differences between the X-ray structure and the Head portion of the Mediator cryo-em map most likely reflect the effect of variability of Head module conformation on the EM map: the Mediator particles that used to calculate the cryo-em map displayed significant conformational variability 9. Med17 is shown in blue, Med11 in purple, Med22 in dark green, Med6 in yellow, Med8 in red, Med18 in orange, and Med20 in green $'

27 RESEARCH a! b! Med22! N86K! Med17! G353C! Med6! Q49L! Med6! R132G! Med6! F194L! Med18! T22I! Med6! I68L! Med6! L94P! Med6! F125Y! Med20! P14H! c! Med11! T47A! Supplementary Figure 16 Mapping of genetic mutations on the Head structure. (a) The srb suppressor mutations are shown within the Mediator Head module structure (Med17: Srb4, Med18: Srb5, Med20: Srb2, Med22: Srb6). Three mutants (Med18 T22I, Med17 G353, and Med22 N86K) are found in the central Joint, and Med20 P14H is located in the Movable jaw. (b) Mapping med6 temperature sensitive (ts) mutations 46. A total of 6 med6 ts mutations (Gln 49 Leu, Ile 68 Leu, Leu 94 Pro, Phe 125 Tyr, Arg 132 Gly, and Phe 192 Leu) are indicated within Med6 47 subunit. Corresponding residues in the model are colored in red, or, alternatively, approximate positions of residues are indicated by circles. (c) Mapping of Med11 residue 47 (T) in the Head module structure. Med17 is colored blue, Med11 purple, Med22 dark green, Med6 yellow, Med8 red, Med18 cyan, and Med20 orange. $( 27

28 RESEARCH Mediator! Pol II! Med6! Rpb4! Rpb7! Head Module! Supplementary Figure 17 Model of the Head-Pol II. Docking of our X-ray crystal structure into the Head portion of the Mediator-Pol II holoenzyme EM map shows excellent agreement and results in a unique fit. Med17 is shown in blue, Med11 in purple, Med22 in dark green, Med6 in yellow, Med8 in red, Med18 in cyan, and Med20 in orange. $) 28

29 RESEARCH Mediator! Head! Rpb1! CTD phosphorylation (a.u.)! Supplementary Figure 18 Stimulation of the CTD kinase of TFIIH by the Mediator Head module. Purified Mediator (0 pmol in lane 1, 0.2 pmol in lane 2, 0.4 pmol in lane 3), and recombinant Head module (0.4 pmol in lane 4, 0.8 pmol in lane 5) was incubated with Pol II (100 ng), TFIIH (15 ng), and!- 32 P labeled ATP at 30 C for 30 min (lanes 1-5) as described 15. CTD phosphorylation was revealed by SDS-PAGE, and a fluorescent image analyzer (top), and was quantified by fluorescent image analysis (bottom). The band due to the Rpb1 subunit of Pol II is indicated. 29

30 RESEARCH a! Med6 F52! Med6 M48! b! Med17 Y269! c! Med17 Y423! Med17 M422! Supplementary Figure 19 Electron density maps showing features of the aromatic residues. The unbiased electron density maps were calculated by SeMet SAD experimental phases. The atomic model is shown in stick representation. The experimental electron density map (blue) is contoured at 1.0 $, and the anomalous map (red) is contoured at 3.0 $. 30

31 " RESEARCH 43 Toth-Petroczy, A. et al. Malleable machines in transcription regulation: the mediator complex. PLoS Comput Biol 4, e (2008). 44 Baumli, S., Hoeppner, S. & Cramer, P. A conserved mediator hinge revealed in the structure of the MED7.MED21 (Med7.Srb7) heterodimer. J Biol Chem 280, (2005). 45 Han, S. J. et al. Activator-specific requirement of yeast mediator proteins for RNA polymerase II transcriptional activation. Mol Cell Biol 19, (1999). 46 Lee, Y. C. & Kim, Y. J. Requirement for a functional interaction between mediator components Med6 and Srb4 in RNA polymerase II transcription. Mol Cell Biol 18, (1998). 47 Koh, S. S., Ansari, A. Z., Ptashne, M. & Young, R. A. An activator target in the RNA polymerase II holoenzyme. Mol Cell 1, (1998). " 31