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Core Mediator structure at 3.4 Å extends model of transcription initiation complex


Mediator is a multiprotein co-activator that binds the transcription pre-initiation complex (PIC) and regulates RNA polymerase (Pol) II1,2,3. The Mediator head and middle modules form the essential core Mediator (cMed)4,5,6, whereas the tail and kinase modules play regulatory roles7. The architecture of Mediator5,8,9,10 and its position on the PIC5 are known, but atomic details are limited to Mediator subcomplexes11,12. Here we report the crystal structure of the 15-subunit cMed from Schizosaccharomyces pombe at 3.4 Å resolution. The structure shows an unaltered head module13,14,15, and reveals the intricate middle module, which we show is globally required for transcription. Sites of known Mediator mutations cluster at the interface between the head and middle modules, and in terminal regions of the head subunits Med6 (ref. 16) and Med17 (ref. 17) that tether the middle module. The structure led to a model for Saccharomyces cerevisiae cMed that could be combined5 with the 3.6 Å cryo-electron microscopy structure of the core PIC (cPIC)18. The resulting atomic model of the cPIC–cMed complex informs on interactions of the submodules forming the middle module, called beam, knob, plank, connector, and hook. The hook is flexibly linked to Mediator by a conserved hinge19 and contacts the transcription initiation factor IIH (TFIIH) kinase that phosphorylates the carboxy (C)-terminal domain (CTD) of Pol II and was recently positioned on the PIC20. The hook also contains residues that crosslink to the CTD and reside in a previously described cradle5. These results provide a framework for understanding Mediator function, including its role in stimulating CTD phosphorylation by TFIIH.

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Figure 1: Crystal structure of S. pombe cMed.
Figure 2: Submodule architecture of cMed.
Figure 3: Model of S. cerevisiae cMed and sites of mutations.
Figure 4: Model of the S. cerevisiae transcription initiation complex.

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We thank members of the Cramer laboratory, in particular C. Plaschka, S. Sainsbury, C. Engel, L. Wenzeck, and L. Larivière. We thank K. Maier and P. Rus for 4-thiouracil sequencing (4tU-seq) data acquisition, and S. Berres and B. Schwalb for bioinformatics. We thank C.-T. Lee and H. Urlaub for mass spectrometry. We thank the staff at EMBL beamline P14 at the Deutsches Elektronen Synchrotron, in particular G. Bourenkov, and on beamline PXI at the Swiss Light Source in Villigen, Switzerland, in particular T. Tomizaki. K.N. was supported by postdoctoral fellowships from the Human Frontier Science Program (LT000621/2012-L), the Uehara Memorial Foundation and the Japan Society for the Promotion of Science. P.C. was supported by the Deutsche Forschungsgemeinschaft (SFB860, SPP1935) and the European Research Council Advanced Investigator Grant TRANSREGULON (grant agreement number 693023).

Author information

Authors and Affiliations



K.N. performed experiments and data analysis. T.S. assisted with diffraction data collection. P.C. designed and supervised research. K.N. and P.C. interpreted the data and wrote the manuscript.

Corresponding author

Correspondence to Patrick Cramer.

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The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks S. Hahn, A. Leschziner and D. Taatjes and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 cMed structure determination.

a, Schematic of the expression vector design for co-expression of the S. pombe cMed subunits. We co-expressed Med1 with the other 15 cMed subunits to obtain higher solubility and yield, but Med1 was dissociating during crystallization and is not present in the cMed structure. Details on the vectors are available upon request. b, SDS–polyacrylamide gel electrophoresis analysis of purified recombinant 16-subunit S. pombe cMed. Head and middle module subunits are labelled in light blue and violet, respectively. Asterisks mark essential subunits. The identity of the bands was verified by mass spectrometry. c, Quality of the electron density map. Depicted is the 2Fo − Fc electron for a helical element in the hook with the refined atomic structure superimposed. The density was calculated from the refined model and contoured at 1.0σ. d, Superposition of four anomalous difference electron density maps, contoured at 3σ, revealing selenium peaks that indicate the position of methionine residues. A total of 29 additional methionine residues were introduced in three mutant variants of cMed. The large number and good distribution of sequence markers facilitated modelling and led to a refined atomic model of very high confidence.

Extended Data Figure 2 Structure of the Mediator middle module.

a, Ribbon model of the middle module as observed within the cMed crystal structure. b, The eight middle module subunits were separated to reveal details of their structure and secondary structure elements. Numbering of secondary structure elements is consistent with our previous Mediator subcomplex crystal structures12.

Extended Data Figure 3 Knob orientation and CTD binding.

Compared with our cMed crystal structure (top), the knob submodule in a previous model based on low-resolution EM and crosslinking20 (bottom) is rotated by ~180°, probably because structural restraints were not sufficient to define its orientation. The knob is not involved in crystal contacts in our cMed structure, and thus its position is not influenced by crystal packing. S. c., S. cerevisiae; S. p., S. pombe.

Extended Data Figure 4 Mediator head–middle module interface.

a, Location of the four contact sites (interfaces I–IV) between the Mediator head and middle modules and the two tethers between the head and middle modules indicated on the cMed crystal structure. b, Detailed views of the four interfaces I–IV. The direction of view relative to the structure in a is provided in the upper left corner of each view. Prominent interacting elements and residues are labelled. Labels for selected mutated residues (corresponding to S. cerevisiae) are encircled. The close-up view of interface III shows potential clashes with a CTD peptide (magenta) present in a previous S. cerevisiae head module–CTD peptide complex15 after superposition onto the cMed head module. c, Close-up views of the Med6C and Med17N tethers, their interactions, and sites of selected mutations. d, Mutations in the middle module lead to global decrease in RNA synthesis activity in the yeast S. cerevisiae. RNA synthesis rates were obtained by 4tU-seq using a 5-min pulse of metabolic labelling with 4-thiouracile. Data represent the average of two biological replicates. The strains analysed here were from ref. 44. The EWE3 strain carries a 25-residue C-terminal deletion from Med7. The EWE4 strain has a point mutation in Med21 (L76P). The EWE5 strain has a 34-residue C-terminal truncation in Med10. As parental strain, we used EWE+ (MATa ade- can1-100 cyh2r his3-11,15 leu2-3,112 trp1-1 ura3 hsp82- DHSE-lacZ).

Extended Data Figure 5 Modelling transcription initiation complexes.

a, Fit of S. cerevisiae cMed model into our previous cryo-EM density of the cPIC–cMed complex5. The density at 9.7 Å resolution is shown as a transparent surface and the cPIC model and the structure-derived S. cerevisiae cMed model are shown as ribbon representation. See Supplementary Fig. 5 for sequence alignments used to construct the cMed homology model. b, B-factor distribution of the S. pombe cMed structure reveals high degrees of flexibility at the interface between the head and middle modules, most notably at the shoulder, hinge, and connector. c, Two views of the cPIC–cMed model. Compared with the model in a, the minimal cPIC was replaced by the 3.6 Å cryo-EM structure of cPIC18 that comprises also TFIIA and TFIIE. Crosslink positions5 between the middle module and Pol II are shown as red spheres. d, Close-up views of interface C. The hydrophilic patch of α1 in Med4 and α2 in Med9 are facing the Pol II foot, which explains two known protein crosslinks5. e, Superposition of the cPIC–cMed model shown in b onto the published yeast (S. cerevisiae (S.c.))29 and human (H.s.)30 PIC cryo-EM densities visualizes the TFIIH core including Rad3, which is approached by Med10, Med14N, and Med19 in the hook submodule.

Extended Data Figure 6 Comparison of the middle module from our cMed crystal structure with a recent model derived from cryo-EM.

a, Ribbon representation of the middle module from our cMed crystal structure reported here. Subunits are coloured as in Fig. 1. b, Middle module taken from a model derived recently from cryo-EM at up to 4.4 Å resolution31 (PDB accession number 5U0P). To allow for comparison of subunit assignment, subunits assigned by ref. 31 are coloured using the colour key of Fig. 1. c, Comparison of the structure in a (this work) with the EM-based model in b (ref. 31, PDB accession number 5U0P) reveals differing and undefined regions in the latter model31. The EM-based model31 is shown as in b but with regions that differ from our crystal structure shown in different colours. Compared with our crystal structure (this work), the EM-derived model31 contains regions where subunits or parts of subunits were assigned differently (red), regions where the amino-acid sequence remained unassigned (orange), and regions where the register of the amino-acid sequence was shifted (green, ‘out-of-register’ regions). Whereas the beam submodule is largely correct in the EM-derived model31, the hook structure was not defined. In the knob, the peripheral, newly built regions were assigned to different subunits or shifted in register in the EM-derived model31. In the plank, subunits were correctly assigned but the amino-acid register was not assigned in the EM-derived model31. In more detail, middle module subunits in the EM-derived model31 differ from our crystal structure as follows. In Med4, Med4C (100–120, 139–173, 197–211) is lacking, one of the Med4 knob helices was assigned differently (to subunit Med14), and the rest of Med4 was built as backbone. In Med9, the amino-acid register is shifted by ~20–30 residues throughout. Med10 entirely deviates from our crystal structure except for one helix with unassigned register. For Med14, Med14N (1–114) is lacking, and ~50 C-terminal residues are apparently erroneous (the modelled Med14 C-terminal residues seem to belong to the beam submodule and not to the C-terminal part of Med14, and residues 880–901 that have been modelled do not exist in the S. pombe Med14 sequence according to UniProtKB database entry Q9P7Y4, MED14_SCHPO). Med19 entirely differs from our crystal structure and has been modelled into densities belonging to Med10 and Med14N.

Extended Data Table 1 X-ray data collection and refinement statistics
Extended Data Table 2 X-ray data collection statistics for SeMet-labelled crystals of methionine mutant cMed variants
Extended Data Table 3 Mutations in the Mediator middle module and selected mutations in head subunits that were not previously analysed structurally5

Supplementary information

Supplementary Information

This file contains Supplementary Figure 1, sequence alignments for middle module subunits. (PDF 376 kb)

Supplementary Data

This file contains the S. cerevisiae cMed model. (TXT 1047 kb)

Supplementary Data

This file contains the S. cerevisiae cPIC-cMed model. (TXT 4362 kb)

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Nozawa, K., Schneider, T. & Cramer, P. Core Mediator structure at 3.4 Å extends model of transcription initiation complex. Nature 545, 248–251 (2017).

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