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Structure of promoter-bound TFIID and model of human pre-initiation complex assembly

Nature volume 531, pages 604–609 (2016)Cite this article

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A Corrigendum to this article was published on 20 April 2016

This article has been updated

Abstract

The general transcription factor IID (TFIID) plays a central role in the initiation of RNA polymerase II (Pol II)-dependent transcription by nucleating pre-initiation complex (PIC) assembly at the core promoter. TFIID comprises the TATA-binding protein (TBP) and 13 TBP-associated factors (TAF1–13), which specifically interact with a variety of core promoter DNA sequences. Here we present the structure of human TFIID in complex with TFIIA and core promoter DNA, determined by single-particle cryo-electron microscopy at sub-nanometre resolution. All core promoter elements are contacted by subunits of TFIID, with TAF1 and TAF2 mediating major interactions with the downstream promoter. TFIIA bridges the TBP–TATA complex with lobe B of TFIID. We also present the cryo-electron microscopy reconstruction of a fully assembled human TAF-less PIC. Superposition of common elements between the two structures provides novel insights into the general role of TFIID in promoter recognition, PIC assembly, and transcription initiation.

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Figure 1: Cryo-EM reconstruction of the TFIID-IIA–SCP complex.
Figure 2: A TAF1–TAF7 subcomplex forms a downstream promoter-binding module.
Figure 3: TAF2 APD.
Figure 4: Structural TAFs within lobe C.
Figure 5: Model of the TFIID-based PIC.

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Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

Cryo-EM density maps have been deposited in the Electron Microscopy Data Bank (EMDB) under codes EMD-3304 (TFIID-IIA–SCP complex), EMD-3305 (locally refined BC-core of TFIID-IIA–SCP complex), EMD-3306 (locally refined lobe C of TFIID-IIA–SCP complex), and EMD-3307 (TAF-less PIC). Model coordinates have been deposited in the Protein Data Bank (PDB) under accession number 5FUR.

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Acknowledgements

We thank P. Grob, S. Howes, and R. Zhang for electron microscopy support; T. Houweling for computer support; S. Scheres for technical advice on image processing; A. Patel for discussion; C. Inouye for providing us with recombinant TFIIF; S. Zheng for providing TAF4 mAb; and D. King for providing peptides. We are thankful to R. Tjian and J. Kadonaga for their comments on the manuscript. Computational resources were provided in part by the National Energy Research Scientific Computing Center (DE-AC02-05CH11231). This work was funded by NIGMS (GM63072 to E.N.) and Spanish Ministry of Economy and Competitiveness (BFU2013-44306P to P.C.). R.K.L. was supported by the NIGMS Molecular Biophysics Training Grant (GM008295). E.N. is a Howard Hughes Medical Institute Investigator.

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Author notes

  1. Yuan He

    Present address: †Present address: Interdisciplinary Biological Sciences Program, Northwestern University, Evanston, Illinois 60208, USA.,

Authors and Affiliations

  1. Biophysics Graduate Group, University of California, Berkeley, 94720, California, USA

    Robert K. Louder

  2. Department of Molecular and Cell Biology, QB3 Institute, University of California, Berkeley, 94720, California, USA

    Yuan He & Eva Nogales

  3. Molecular Biophysics and Integrative Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    Yuan He & Eva Nogales

  4. Department of Biological Physical Chemistry, Rocasolano Physical Chemistry Institute, CSIC, Serrano 119, Madrid, 28006, Spain

    José Ramón López-Blanco & Pablo Chacón

  5. Howard Hughes Medical Institute, University of California, Berkeley, 94720, California, USA

    Jie Fang & Eva Nogales

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Contributions

R.K.L. and Y.H. designed and performed the experiments; J.R.L.-B. and P.C. performed structural modelling; J.F. purified the TFIID, Pol II, TFIIE, and TFIIH; R.L., Y.H., and E.N. analysed the data and wrote the paper.

Corresponding author

Correspondence to Eva Nogales.

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Extended data figures and tables

Extended Data Figure 1 Cryo-EM of the TFIID-IIA–SCP complex.

a, Representative micrograph of frozen-hydrated TFIID-IIA–SCP complexes. Examples of particle picks are indicated by the green circles; 203,163 such picks were made from 1,253 total micrographs. b, Initial classification and refinement scheme for the TFIID-IIA–SCP structure (see Methods). c, Idealized dose-dependent B-factor plot based on cryo-EM data collected on microtubules under similar imaging conditions. This plot was used for the particle polishing step in b. d, e, Fourier shell correlation plot (d) and local resolution estimation (e) for the final reconstruction shown in b.

Extended Data Figure 2 Focused classification and refinement of the promoter-bound BC-core and lobe C of TFIID.

a, b, Scheme for focused classification and refinement of the BC-core region (a) or lobe C region of the TFIID-IIA–SCP structure (b) (see Methods). c, d, Fourier shell correlation plots (c) and local resolution estimations (d) of the BC-core and lobe C maps, corresponding to the final structures shown in a and b, respectively. e, Two-dimensional projections of the refined maps for the full TFIID-IIA–SCP structure (left), locally refined BC-core map (middle), and locally refined lobe C map (right). The maps used to calculate the projections are the same as the final structures in a, b, and Extended Data Fig. 1b, except that all have been low-pass filtered to 10 Å before calculating projections. f, Three-dimensional classification of 56,457 particles into two classes (solid blue and transparent green), following focused alignment to the lobe C region of the structure. The resulting classes have been superposed through their lobe C densities to illustrate the flexibility of lobe B and the upstream region of promoter DNA relative to lobe C and the downstream promoter region. The magnitude of motion within lobe A1 (20 Å) is indicated.

Extended Data Figure 3 Modelling of TBP, TFIIA, and promoter DNA into the cryo-EM density.

a, Previously published reconstructions of TFIID-IIA–SCP in the rearranged state (left; EMDB code 2282) and of free TFIID in the canonical state (right; EMDB code 2287)33. For the former, the densities for TFIIA (orange) and TBP (red) are assigned on the basis of the superposition with the TFIID-IIA–SCP structure from our present study. b, Close-up view of the TBP–TFIIA–TATA module density and fitted structures. The termini of the TBP structure and the three subunits (α, β, and γ) within the TFIIA structure are indicated with circles. In the cell, the α- and β-subunits of TFIIA are translated as a single polypeptide and then are post-translationally cleaved. The location of the long stretch of residues spanning the region between the structured parts of TFIIAα and TFIIAβ (TFIIAαβ 52–329) is indicated as a dashed line. Note that only 34 of the residues within this flexible loop (52–58 and 303–329) are included in the TFIIA construct used for this study. Mutational analysis in yeast has shown that mutation of an isoleucine residue (I23 in humans, I27 in yeast; represented in green spheres) to lysine at the tip of the TFIIA four-helix bundle disrupts the interaction between TFIID and TFIIA35. c, Mapping of the MPE.Fe(II) cleavage pattern for SCP DNA bound to TFIID-IIA, on the basis of data published in ref. 33. d, Mapping of the downstream core element (DCE)9 sequence onto the SCP DNA within the TFIID-IIA–SCP structure from our present study.

Extended Data Figure 4 Structural modelling and conservation of the TAF1 promoter-binding domains.

a, TAF1 WH domain (grey) in complex with promoter DNA (cyan) superposed with the DNA-binding WH domain of the transcription factor E2F4 (PDB accession number 1CF7, magenta) in complex with its cognate DNA, with the alignment based on the protein (left) or DNA (right) components. b, Sequence alignment and secondary structure map of the TAF1 WH domain, used to calculate the conservation scores depicted in Fig. 2c (Hs, Homo sapiens; Dr, Danio rerio; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans; At, Arabidopsis thaliana; Sp, Schizosaccharomyces pombe; Sc, Saccharomyces cerevisiae). The conserved positively charged residues that are in close proximity to the promoter DNA within the docked structure (K818, R864, K865, K868, and R875) are highlighted in pink. Numbering is based on the human sequence. c, Sequence alignment of a region of the TAF1 DUF3591 corresponding to the internal segment that is missing from the crystal structure and neighbouring residues. The putative Inr-binding domain (1009–1061) within this segment is highlighted in blue. Numbering is based on the human sequence, and abbreviations are the same as in a. d, Three-dimensional structure prediction for the putative TAF1 Inr-binding domain output by the I-TASSER server67. On the left, the residues are coloured in rainbow from N to C termini, with the terminal residues indicated. On the right, the modelling confidence is depicted in terms of the ResQ score (ribbon colour) and B-factor estimation (ribbon thickness) output by I-TASSER67, with high confidence regions represented by thinner blue ribbon and low-confidence regions represented with thicker red ribbon. e, Secondary structure prediction for the sequence modelled in d (H, helix; C, coil).

Extended Data Figure 5 Structural modelling and conservation of TAF2 APD.

a, Structural arrangement of domains (D1–D4) within the TAF2 APD (bottom) compared with that of human ERAP1 (top, PDB accession number 2YD0)39, a member of the M1 family of aminopeptidases to which TAF2 shares homology. b, Domain arrangement of TAF2, including the four subdomains of the APD (D1–D4), and the C-terminal intrinsically disordered region (IDR). c, Rigid-body docking of the best-conserved domains (D1 and D2) of the homologous human ERAP1 confirms the identity of this density. d, Segmented densities and fitted structures for the four subdomains (D1–D4) of the TAF2 APD. e, Sequence alignment and secondary structure map for the putative DNA-binding regions within domain 3 of the TAF2 APD (species abbreviations are the same as in Extended Data Fig. 4b). Conserved residues that are in close proximity to the DNA within the docked structure are highlighted in pink. The stretch that is depicted as a dashed line shares low sequence similarity with known M1 aminopeptidases. Numbering is based on the human sequence.

Extended Data Figure 6 Structural modelling and conservation of TAF6 and putative TAF8 density.

a, Cryo-EM density of the TAF6 dimer with fitted homology models. Putative regions involved in the homodimer interface are labelled. b, Organization of α-helices within the human TAF6 HEAT-like repeat and unaccounted density (green) around the TAF6 homodimer. c, Sequence alignment and secondary structure map of the TAF6 HEAT repeat domain (species abbreviations are the same as in Extended Data Fig. 4b, except that Al is A. locustae). The green region indicates the region that is unmodelled in our structure, with the two predicted C-terminal helices outlined with dashes. Numbering is based on the human sequence. d, Unaccounted density indicative of two α-helices, located between domain 4 of the TAF2 APD and one copy of the TAF6 HEAT domain, which we attribute to TAF8. e, Sequence alignment of a putative TAF2-interaction domain within TAF8 (species abbreviations are the same as in Extended Data Fig. 4b). The last helix of the structurally determined histone fold domain of TAF8 is depicted in dark blue, while the 26 residue stretch that is predicted to be α-helical is shown in light blue with dashed outline. Secondary structure prediction was performed with PSI-PRED71.

Extended Data Figure 7 Modelling of the TFIID-based PIC.

a, TFIID-based PIC model from Fig. 4, with the density for lobe A2 density (yellow) low-pass filtered to 16 Å and displayed at two different intensity thresholds (lower threshold in transparency). Both thresholds are lower than that used to display the density for the promoter-bound BC-core of TFIID. b, Close-up view of putative interactions between RPB1, -2, and -5 of Pol II and TAF1 of TFIID. c, Comparison of the paths of the promoter DNA within the TFIID-IIA–SCP and TAF-less PIC structures. The promoter DNA from the TFIID-IIA–SCP structure is coloured as in Fig. 1, and the promoter DNA from the TAF-less PIC is coloured in green. View is from the top of the model, relative to a. d, Docking of the core mediator coactivator complex (cMed, EMDB code 2786)46, including the mediator head and middle modules, onto the TFIID-based PIC, on the basis of the structure of a cMED-bound initial transcribing complex. e, Docking of the free yeast mediator complex (brown transparency, EMDB code 2634)47 on the basis of alignment with the core mediator shown in c. Lobe A2 of TFIID (yellow) is depicted similarly as in a.

Extended Data Table 1 Summary of TFIID subunits
Full size table

Supplementary information

Structure of promoter-bound TFIID and insight into human PIC assembly

Cryo-EM structure of human TFIID-IIA-SCP and fitting of atomic models. Superposition of common elements between TFIID-IIA-SCP and the TAF-less PIC leads to a model of a TFIID-based PIC. (MOV 29244 kb)

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Louder, R., He, Y., López-Blanco, J. et al. Structure of promoter-bound TFIID and model of human pre-initiation complex assembly. Nature 531, 604–609 (2016). https://doi.org/10.1038/nature17394

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