The conserved Mediator co-activator complex has an essential role in the regulation of RNA polymerase II transcription in all eukaryotes. Understanding the structure and interactions of Mediator is crucial for determining how the complex influences transcription initiation and conveys regulatory information to the basal transcription machinery. Here we present a 4.4 Å resolution cryo-electron microscopy map of Schizosaccharomyces pombe Mediator in which conserved Mediator subunits are individually resolved. The essential Med14 subunit works as a central backbone that connects the Mediator head, middle and tail modules. Comparison with a 7.8 Å resolution cryo-electron microscopy map of a Mediator–RNA polymerase II holoenzyme reveals that changes in the structure of Med14 facilitate a large-scale Mediator rearrangement that is essential for holoenzyme formation. Our study suggests that access to different conformations and crosstalk between structural elements are essential for the Mediator regulation mechanism, and could explain the capacity of the complex to integrate multiple regulatory signals.
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Structure of mammalian Mediator complex reveals Tail module architecture and interaction with a conserved core
Nature Communications Open Access 01 March 2021
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This work was supported by US National Institutes of Health grants R01 GM67167 (F.J.A.) and R01 GM41628 (R.C.C. and J.W.C) and by a grant to the Stowers Institute from the Helen Nelson Medical Research Fund at the Greater Kansas City Community Foundation. We thank C. Gustafsson for providing the Med7-TAP, Med13Δ S. pombe strain used for the high-resolution EM analysis. S.G. contribution will fulfil, in part, requirements for her PhD thesis research as a student registered with the Open University.
The authors declare no competing financial interests.
Reviewer Information Nature thanks S. Hahn, A. Leschziner and D. Taatjes for their contribution to the peer review of this work.
Extended data figures and tables
a, Localization of ScMED subunits. b, Coomassie blue-stained SDS–PAGE analysis (4–20% gradient gel) of purified wild-type (WT), ΔMed2 and ΔMed27 SpMED. The Med2 band is comparatively weaker, suggesting that the subunit might be substoichiometric in purified wild-type SpMED. Deletion of Med2 results in loss of Med27, but not the other way around. The band marked by the asterisk corresponds to Rpb1, which was confirmed by mass spectrometry. Subunits are coloured by module (head in pink; middle in blue; tail in grey). c, 2D class averages for wild-type, ΔMed2 and ΔMed27 SpMED, and colour-coded (by standard deviation values from the average) difference maps indicating the position of Med2 and Med27 (highlighted by yellow arrowheads). d, Wild-type SpMED 2D class averages and a close-up showing a Med27–tail connection (bottom-right arrowhead) comparable to the connection between the Med4–Med9 four-helix bundle and the rest of the middle module (top-left arrowhead). e, A raw micrograph showing a typical area of a SpMED cryo-EM sample.
Extended Data Figure 2 2D clustering, 3D classification, and refinement of Mediator and holoenzyme images recorded from SpMED cryo-EM samples.
a, Particle images were clustered into homogeneous classes after automated picking. Clustering and alignment showed that a fraction (29%, green box) of particle images initially picked as possible ‘holoenzyme’ correspond to images of particles in which RNAPII, loosely tethered to Mediator, dominates alignment and blurs Mediator density (variable Mediator position suggested by pasted semi-transparent Mediator averages). This prevents calculation of reliable 3D maps showing the moving RNAPII bound around the Mediator hook. b, Cryo-EM holoenzyme maps calculated from three different subsets of holoenzyme images all show the same Mediator and RNAPII conformations and relative orientations, with differences limited to the amount of detail apparent in each map. c, SpMED 2D cryo-EM class averages. d, FSC plot and resolution estimation using the gold-standard 0.143 criterion for the SpMED cryo-EM map. e, Angular distribution plot for the SpMED cryo-EM image set. f, Focused refinement (mask indicated by dashed red line) of the SpMED core by masking out the hook, Med1 and tail portions. g, Local refinement of the top portion of the middle module.
a, Head portion of the SpMED cryo-EM map showing secondary structure elements corresponding to individual subunits. The resolution of the head portion of the map is 4.5–5.0 Å. b, Med14 portion of the cryo-EM map and close-ups showing density corresponding to bulky Med14 side chains. The resolution of the Med14 portion of the map is 4.0–4.5 Å. c, Middle portion of the SpMED cryo-EM map showing secondary structure elements corresponding to various subunits, and map segments corresponding to individual middle components. The resolution of the middle portion of the map and its segments is 5–6 Å. d, Cryo-EM structure of the head module. Colours as indicated in Fig. 1c. e, Partial X-ray structure of the head module (PDB code 4H63; left, in grey) compared with the cryo-EM structure of the head module (right), with portions not included in the X-ray crystal structure coloured and labelled. f, Superposition of the X-ray (grey) and cryo-EM (green) structures of the head module shows a close correspondence between common elements, indicating that the overall conformation of the head is not changed by interaction with other Mediator modules. r.m.s.d. values between 744 Cα atom pairs in corresponding portions of the X-ray and cryo-EM structures of the head is 2.1 Å. g, Alignment of Med7 protein sequences from S. pombe and S. cereivisiae. The secondary structure evident in the cryo-EM structure of S. pombe Med7 is indicated. Identical and similar residues are highlighted in green and yellow, respectively. Residues in S. pombe Med7 expected to be part of the hook are highlighted in light blue. h, Cryo-EM structure of the middle module (left) and a predicted model of the middle module41 based on X-ray structures of Med7C–Med21 and Med7N–Med31, homology modelling of Med4–Med9–Med10, and results from XL/MS analysis (right). i, Comparison between S. cerevisiae Med7N–Med21 from the X-ray structure (grey) and S. pombe Med7N–Med21 from the cryo-EM map. j, Fitting of the SpMED middle module structure (solid blue) into the ScMED cryo-EM map (transparent yellow) shows that the middle structure is conserved between S. cerevisiae and S. pombe. k, Comparison between the cryo-EM structure of S. pombe Med7N–Med31 and the X-ray structure of S. cerevisiae Med7N–Med31 shows that this portion of the Mediator structure is highly conserved. The r.m.s.d. value between 84 Cα atom pairs in corresponding portions of the X-ray structure of S. cerevisiae Med7N–Med31 (PDB code 3FBI) and the cryo-EM structure of S. pombe Med7N–Med31 is 2.0 Å.
Extended Data Figure 4 Secondary structure predictions for yeast and human Med14 proteins, and correspondence between published XL/MS analysis of ScMED and the atomic model based on the cryo-EM map of SpMED.
a, b, Secondary structure for S. pombe Med14 (a) and H. sapiens (Hs) Med14 (b). The C-terminal portion of human Med14 (b) is comparatively larger. c, Med17N crosslinks with Med7N–Med21. d, Med14 (RM1) crosslinks with Med17N. e, Med6C crosslinks with Med14 (RM1). f, Med14 (RM1) crosslinks with Med17 (Med17 jaw). The residue number/ranges shown correspond to SpMED subunits. For all residue pairs in the SpMED atomic model corresponding to crosslinked residues in the XL/MS analysis of ScMED, the distance between residues in a pair is below 35 Å (the maximum expected distance between crosslinked residues based on the structure of the crosslinking reagent).
Extended Data Figure 5 Cryo-EM analysis of the S. pombe holoenzyme and comparison with the S. pombe RNAPII X-ray structure (PDB code 3H0G) and S. cerevisiae core MED-ITC and MED-PIC cryo-EM maps.
a, 2D holoenzyme cryo-EM class averages. b, FSC plot for the holoenzyme cryo-EM map. c, Angular distribution plot for the holoenzyme cryo-EM map. d, Local resolution values in the holoenzyme cryo-EM map. e, The Rpb1 foot portion of the RNAPII X-ray structure (PDB code 3H0G), and neighbouring domains, fitted into the holoenzyme map. f, The Rpb3/Rpb11 portion of the RNAPII X-ray structure (PDB code 3H0G), and neighbouring domains, fitted into the holoenzyme map. g, A slice through the central portion of a front view of the RNAPII crystal structure (PDB code 3H0G) shows the bridge helix and comparatively weak Rpb4/Rpb7 density making contacts with Med8 and Med17 in the head module. h, A slice through the central portion of a top view of the RNAPII X-ray structure (PDB code 3H0G) shows Rpb4/Rpb7 contacting Med8 and Med17. i, S. pombe holoenzyme segmented into modules. j, S. cerevisiae core MED-ITC with modules from S. pombe holoenzyme fitted in. The gap between Med4–Med9 and the Rpb1 foot in S. cerevisiae core MED-ITC is hidden by the fitted S. pombe holoenzyme middle module. k, S. cerevisiae MED-PIC (EMDB-8307) with modules from S. pombe holoenzyme fitted in. There is no gap between Med4–Med9 and the Rpb1 foot in S. cerevisiae MED-PIC.
Extended Data Figure 6 Cryo-EM map and model of the Mediator–RNAPII holoenzyme, and comparison with the S. cerevisiae core MED-ITC cryo-EM map (EMDB-2786).
a, Cryo-EM map of the holoenzyme at 7.8 Å resolution and corresponding atomic model (head in magenta; middle in blue; Med14 in green; RNAPII in yellow). Contacts between Mediator and RNAPII highlighted by squares. b, Rpb3/Rpb11 interaction with Med20 in the movable jaw of the head module. The Rpb3 C92 residue, important for the RNAPII interaction with Mediator, is indicated. Med20 and Rpb3 are shown in purple and red, respectively. c, Putative CTD density interacting between the neck of the head module and knob of the middle module. Main-chain CTD residues (Y10–Y24) are shown in cyan after docking the crystal structure of an ScMED head–CTD complex (PDB code 4GWQ) into the head portion of the holoenzyme cryo-EM map. d, Rpb1 foot interaction with Med4N and Med9N in the middle module. Selected Rpb1 residues are indicated for reference. e, The Mediator–RNAPII holoenzyme complex (EMDB-2786) segmented into head module, middle module, Med14, general transcription factors, and promoter DNA. f, Fitting of S. pombe holoenzyme Med14 subunit, and head and middle modules (without any changes to relative module orientation) into the Mediator portion of the EMDB-2786 map. g, Comparison of the RNAPII positions in the S. pombe holoenzyme and the Mediator–RNAPII cryo-EM maps (EMDB-2786) after matching of the head modules (EMDB-2786 head module and RNAPII in purple and orange, respectively; S. pombe holoenzyme RNAPII in light green). Mediator and RNAPII portions of both complexes match, but there is a difference in the RNAPII rotation. h, Comparison of the Med20–Rpb3 contact in the S. pombe holoenzyme and EMDB-2786 after matching of Med20 subunits (EMDB-2786 Med18/20 and Rpb3 subunits in purple and light orange, respectively; S. pombe holoenzyme Med18, Med20 and Rpb3 subunits in light blue, khaki and red, respectively). i, Comparison of putative CTD density in S. pombe holoenzyme map (left; density in gap between the neck of the head module and knob of the middle module), with the position of the CTD peptide in an X-ray structure of a head–CTD complex (PDB code 4GWQ; molecular model of the head in 4GWQ in light yellow, CTD peptide density in cyan). The N-terminal domain of Med6 from the 4GWQ structure (amino acids 1–120) was not considered for head module alignment, because the S. cerevisiae Med6 has a 42-amino-acid insertion starting at residue 65. j, Comparison of head–Rpb4/Rpb7 contacts (SpMED head module in pink as molecular model). k, Comparison of Med4/Med9–Rpb1 foot contacts (SpMED middle module in light blue as molecular model).
Extended Data Figure 7 Conserved conformation of head, middle and Med14–middle interface between S. pombe Mediator and S. pombe holoenzyme, and Mediator rearrangements upon holoenzyme formation.
a, The structure of the head module is very similar in free Mediator (yellow) and holoenzyme (blue), except for the positions of the Med17N and Med6C portions, which connect to the knob and Med14, respectively. r.m.s.d. value between 1,334 Cα atom pairs in corresponding portions of the Mediator and holoenzyme EM structures of the head module is 1.9 Å (the Med17N and Med14C domains that move with the knob were not included in the r.m.s.d. calculation). b, The structure of the middle module is also very similar in free Mediator (yellow) and holoenzyme (blue). The r.m.s.d. value between 801 Cα atom pairs in corresponding portions of the Mediator and holoenzyme cryo-EM structures of the middle module is 2.7 Å. c, Med14 has a considerable conformational change between free Mediator (yellow) and holoenzyme (blue). d, The structure of the Med14 (RM1)–middle interface is the same in free Mediator and holoenzyme. e, Med14-interacting Med17N and Med6C domains move along with the top of Med14. f, Repositioning of the middle module upon holoenzyme formation closes the CTD-binding gap. It also brings the middle module Med4 and Med9 subunits into contact with the Rpb1 foot of the polymerase (highlighted in light green). The rigidity of the middle module effectively links the contacts Mediator establishes with the CTD and foot domains of Rpb1. g, S. cerevisiae core MED-ITC structure with the head, middle and Med14 portions coloured in purple, blue and green, respectively. h, Matching the head portion of S. cerevisiae core MED-ITC to the head portion of the published cryo-EM map of free ScMED1 (semi-transparent teal) shows a large rearrangement of the middle module upon ScMED interaction with RNAPII. i, Matching the head portion of S. cerevisiae MED-PIC to the head portion of the published cryo-EM map of free ScMED1 (semi-transparent teal) also shows a large rearrangement of the middle module upon ScMED interaction with RNAPII.
a, Left, Coomassie blue-stained SDS–PAGE analysis (4–20% gradient gel) of purified wild-type, and ΔMed31 ScMED. Right, effect of Med31 deletion (ΔMed31) on the interaction of ScMED with RNAPII. Wild-type and ΔMed31 ScMED were purified by TAP-tagging. Protein elutes from a calmodulin resin were analysed. b, The X-ray structure of S. cerevisiae Med7N/31 (PDB code 3FBI) was fitted into the S. pombe holoenzyme cryo-EM map (S. cerevisiae Med31 in green and Med7N in orange). ScMED Med31 residues expected to be in close contact with the CTD were mutated as indicated. Head, middle and Med14 portions in the S. pombe holoenzyme cryo-EM map are shown in transparent pink, cyan and green, respectively. c, Med31 sequence conservation in S. cerevisiae, S. pombe and human (Hs) Mediators. d, Effect of Med31 point mutations on the Mediator interaction with RNAPII. Med31 mutations Y41E and T44P resulted in a considerable decrease in the RNAPII interaction. Mutation Y38A had a smaller but still considerable effect. The Q45A/Q46A double mutation had no effect on RNAPII binding. A K83A mutation of a lysine located far from the CTD also had no effect on the RNAPII interaction. e, 2D class averages showing that Med31 point mutations that affect RNAPII interactions do not result in destabilization of the knob (knob highlighted by yellow arrowhead in magnified insets). Wild-type and ΔMed31 class averages shown for comparison.
Extended Data Figure 9 Variability in conformation of PIC particles and cryo-EM map of a TFIIK-containing PIC.
a, PIC maps showing various degrees of TFIIH organization. b, Cryo-EM map of a TFIIK-containing PIC. TFIIH and TFIIK are coloured in yellow and red, respectively. c, 2D cryo-EM class averages of TFIIK-containing PIC. d, Left, FSC plot for the TFIIK-containing PIC cryo-EM map (the resolution was estimated using the gold-standard 0.143 criterion). Right, angular distribution plot for the TFIIK-containing PIC cryo-EM map. e, Comparison between projections of the TFIIH portion of the TFIIK-containing PIC map calculated from cryo-EM data (left), and 2D class averages calculated from negative-stained images of TFIIH–TFIIK (right). In TFIIH–TFIIK 2D class averages, TFIIK can be found in three positions, one of which matches the one observed in the TFIIK-containing PIC map. f, Possible model for TFIIK-containing PIC interaction with the holoenzyme conformation of Mediator. The position of the mobile tail module is indicated by the dashed circle.
This file contains Supplementary Text and Supplementary References. (PDF 119 kb)
Identification of S. pombe Mediator subunits in Med4-FLAG, Med7-FLAG, and δMed13 Med7-TAP SpMED preparations by MudPIT mass spectrometry. This table shows the number of peptides (P) and spectra (S), sequence coverage (SC, as%) and distributed normalized spectral abundance factor (dNSAF; number of spectra, normalized to protein length and total number of spectra detected in run) for each Mediator subunit copurifying with S. pombe FLAG- or TAP-tagged Med2, Med4, and Med7. Control samples are FLAG-immunopurified proteins from the standard parental 972h- strain or TAP-purified proteins from strain TP161. The number of spectra corresponding to a particular protein identified in MudPIT runs varies with the protein's length and relative abundance. dNSAF values provide a measure of the relative amount of the same protein across a number of different samples, but only a rough estimate of the relative amounts of each protein detected in a MudPIT data set. (XLSX 33 kb)
Atomic model of SpMED (MOV 6209 kb)
Atomic model of the SpMED-RNAPII holoenzyme (MOV 6383 kb)
Morphing between the free and holoenzyme conformations of SpMED (MP4 1037 kb)
Morphing between the free and holoenzyme conformations of SpMED and how it changes interaction with the PIC.
PIC map is shown in transparent gray, with RNAPII in orange, TFIIH in yellow, and TFIIK in pink. (MP4 695 kb)
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Tsai, KL., Yu, X., Gopalan, S. et al. Mediator structure and rearrangements required for holoenzyme formation. Nature 544, 196–201 (2017). https://doi.org/10.1038/nature21393
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