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Structure of the human Mediator–RNA polymerase II pre-initiation complex

Abstract

Mediator is a conserved coactivator complex that enables the regulated initiation of transcription at eukaryotic genes1,2,3. Mediator is recruited by transcriptional activators and binds the pre-initiation complex (PIC) to stimulate the phosphorylation of RNA polymerase II (Pol II) and promoter escape1,2,3,4,5,6. Here we prepare a recombinant version of human Mediator, reconstitute a 50-subunit Mediator–PIC complex and determine the structure of the complex by cryo-electron microscopy. The head module of Mediator contacts the stalk of Pol II and the general transcription factors TFIIB and TFIIE, resembling the Mediator–PIC interactions observed in the corresponding complex in yeast7,8,9. The metazoan subunits MED27–MED30 associate with exposed regions in MED14 and MED17 to form the proximal part of the Mediator tail module that binds activators. Mediator positions the flexibly linked cyclin-dependent kinase (CDK)-activating kinase of the general transcription factor TFIIH near the linker to the C-terminal repeat domain of Pol II. The Mediator shoulder domain holds the CDK-activating kinase subunit CDK7, whereas the hook domain contacts a CDK7 element that flanks the kinase active site. The shoulder and hook domains reside in the Mediator head and middle modules, respectively, which can move relative to each other and may induce an active conformation of the CDK7 kinase to allosterically stimulate phosphorylation of the C-terminal domain.

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Fig. 1: Structure of the human Mediator–PIC complex.
Fig. 2: Features of the structure of human Mediator.
Fig. 3: Interactions between Mediator and the core PIC.
Fig. 4: Interactions between Mediator and TFIIH kinase.

Data availability

The cryo-EM density reconstructions and models were deposited with the Electron Microscopy Data Bank (EMDB) (accession codes EMD-12609 for Mediator in complex with the Pol II stalk and EMD-12610 for Mediator in complex with the PIC) and with the Protein Data Bank (PDB) (accession code 7NVR). All data are available in the Article or its supplementary files. Source data are provided with this paper.

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Acknowledgements

We thank past and present members of the P.C. laboratory, particularly U. Neef, P. Rus and F. Grabbe for maintenance of the insect cell culture and purification of the recombinant human initiation factors; and we thank C. Dienemann and U. Steuerwald for maintenance of the cryo-EM facility. S.R. was supported by a Peter-and-Traudl-Engelhorn postdoctoral fellowship; S.A. was supported by an H2020 Marie Curie Individual Fellowship (894862); and P.C. was supported by the Deutsche Forschungsgemeinschaft (EXC 2067/1 39072994, SFB860 and SPP2191) and the ERC Advanced Investigator Grant CHROMATRANS (grant agreement no. 882357).

Author information

Affiliations

Authors

Contributions

S.R. performed all experiments and data analysis, except for the following: S.S. established the human PIC preparation; S.A. provided human PIC coordinates; S.A. and S.S. assisted with data processing and structural modelling; and C.D. assisted with cryo-EM grid preparation and data collection. P.C. designed and supervised research. S.R., S.S., S.A. and P.C. interpreted the data and wrote the manuscript.

Corresponding author

Correspondence to Patrick Cramer.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Steve Hahn and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Preparation of the human Mediator–PIC complex.

a, Size-exclusion chromatography of recombinant human Mediator shows a single peak. SDS–PAGE analysis (replicated three times) shows the presence of 20 Mediator subunits, confirmed by mass spectrometry (not shown). Raw SDS–PAGE data are provided in Supplementary Fig. 2. b, SDS–PAGE analysis of the human Mediator–PIC complex after isolation from a sucrose gradient (replicated three times). Raw SDS–PAGE data are provided in Supplementary Fig. 2. c, Recombinant 20-subunit human Mediator is able to stimulate Pol II CTD phosphorylation by CDK7 in the presence of ATP. Pol II CTD phosphorylation was assessed by western blotting against phosphorylated Ser5 of the CTD heptad repeat. An antibody against RPB3 was used to obtain the loading control. Experiments were performed in triplicate (P1–P3 and M1–M3) and a negative control sample without ATP (PC and MC) was included to exclude prior CTD phosphorylation. The bar diagram illustrates an around 4.5-fold stimulation of Pol II CTD phosphorylation in the Mediator–PIC samples over the PIC samples. Data are mean ± s.d. of three independent experiments (replicated three times). The mean value of the triplicates was used as the centre measure for error bars (mean = 4.47). Statistical significance with a P value of 3.43 × 10−6 was determined using a one-tailed unpaired t-test (***P < 0.001, **P < 0.01, *P < 0.05). Raw data for the western blots are available in Supplementary Fig. 2.

Source data

Extended Data Fig. 2 Cryo-EM data processing.

a, Representative cryo-EM micrograph of the human Mediator–PIC complex (replicated more than 20,000 times). Scale bar, 300 Å. b, Processing tree describing particle classification. Reconstructions that gave rise to maps used for model building are indicated (blue for focused maps, green for overall maps). Regions corresponding to the core PIC (cPIC), TFIIH (including the CAK), DNA, and Mediator are coloured in grey, pink, dark blue and cyan, and blue, respectively. c, Reconstructions coloured by their local resolution as estimated using RELION. In the angular distribution plots, colour indicates particle representation (white areas indicate unpopulated angles).

Extended Data Fig. 3 FSCs of reconstructions and cryo-EM density.

a, Solvent-corrected ‘gold-standard’ FSCs for the reconstructions shown in Extended Data Fig. 2c. Unmasked (green), masked (blue), and phase-randomized (red) FSCs are also shown. b, Schematic representation of Mediator subunit domain architecture. Regions contributing to submodules are coloured according to the S. pombe cMed structure23. Unassigned regions are coloured grey. c, Local-resolution-filtered map of Mediator–PICwith the fitted structure. The inset shows a magnified view of the CAK module fitting into our density. d, Model-to-map FSCs, showing in blue the fit of the overall structure to the Mediator–PIC and in black the fit of the Mediator–Pol II stalk model to their corresponding maps.

Extended Data Fig. 4 Quality of cryo-EM densities.

Sections of focused-refined Mediator–stalk cryo-EM density overlaid with their respective atomic models. Densities are shown as a blue mesh, and sticks are shown for the model coloured as in Extended Fig. 3c.

Extended Data Fig. 5 Additional structural comparisons.

a, Comparison of the human Mediator–PIC structure (this study) with the free PIC structure25 reveals a different orientation of the Pol II RPB4–RPB7 stalk. The core PIC regions of the Mediator–PIC (in colour) and free PIC (in white) structures were superposed on the 10-subunit Pol II core. Mobile elements in the stalk are indicated and conformational changes between complexes are depicted by red arrows. b, Comparison of the yeast and human Mediator–PIC structures reveals a different relative orientation of the Mediator middle module. The human (in colour) and yeast9 (in white) Mediator–PIC structures were superposed on the well-conserved neck and fixed-jaw domains of the Mediator head module. Conformational changes of Mediator submodules are depicted by red arrows. The proximal tail region of human Mediator was omitted for clarity.

Extended Data Table 1 Cryo-EM data collection and processing

Supplementary information

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Video 1

Structure of the human Mediator-RNA polymerase II pre-initiation complex. Rotating views of the structure. For details refer to text.

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Rengachari, S., Schilbach, S., Aibara, S. et al. Structure of the human Mediator–RNA polymerase II pre-initiation complex. Nature 594, 129–133 (2021). https://doi.org/10.1038/s41586-021-03555-7

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