Structure of the transcription coactivator SAGA

Abstract

Gene transcription by RNA polymerase II is regulated by activator proteins that recruit the coactivator complexes SAGA (Spt–Ada–Gcn5–acetyltransferase)1,2 and transcription factor IID (TFIID)2,3,4. SAGA is required for all regulated transcription5 and is conserved among eukaryotes6. SAGA contains four modules7,8,9: the activator-binding Tra1 module, the core module, the histone acetyltransferase (HAT) module and the histone deubiquitination (DUB) module. Previous studies provided partial structures10,11,12,13,14, but the structure of the central core module is unknown. Here we present the cryo-electron microscopy structure of SAGA from the yeast Saccharomyces cerevisiae and resolve the core module at 3.3 Å resolution. The core module consists of subunits Taf5, Sgf73 and Spt20, and a histone octamer-like fold. The octamer-like fold comprises the heterodimers Taf6–Taf9, Taf10–Spt7 and Taf12–Ada1, and two histone-fold domains in Spt3. Spt3 and the adjacent subunit Spt8 interact with the TATA box-binding protein (TBP)2,7,15,16,17. The octamer-like fold and its TBP-interacting region are similar in TFIID, whereas Taf5 and the Taf6 HEAT domain adopt distinct conformations. Taf12 and Spt20 form flexible connections to the Tra1 module, whereas Sgf73 tethers the DUB module. Binding of a nucleosome to SAGA displaces the HAT and DUB modules from the core-module surface, allowing the DUB module to bind one face of an ubiquitinated nucleosome.

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Fig. 1: Overall structure of SAGA.
Fig. 2: SAGA core-module structure.
Fig. 3: Comparison of the SAGA core module with TFIID.
Fig. 4: Nucleosome binding induces changes in SAGA.

Data availability

The electron density reconstructions and models of the complete SAGA complex, the Tra1 module, the core module, the DUB module–nucleosome complex and the nucleosome-bound state of SAGA were deposited with the Electron Microscopy Data Bank (accession codes EMD-10412, EMD-10413, EMD-10414, EMD-10415 and EMD-10416 respectively) and with the Protein Data Bank (accession codes 6T9I, 6T9J, 6T9K, and 6T9L). All the other relevant data are included in the Supplementary Information or are available from the authors upon request.

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Acknowledgements

We thank M. Ninov for help with mass spectrometry and T. Schulz for yeast fermentation. H.W. was supported by an EMBO long-term fellowship (ALTF 650-2017). H.U. was supported by the Deutsche Forschungsgemeinschaft (SFB860). A.C.M.C. was supported by Wellcome (102535/Z/13/Z). P.C. was supported by the Deutsche Forschungsgemeinschaft (SFB860, SPP1935, EXC 2067/1-390729940), the European Research Council (advanced investigator grant TRANSREGULON, grant agreement no. 693023) and the Volkswagen Foundation.

Author information

H.W. carried out all experiments and data analysis except mass spectrometry analysis. C.D. assisted with cryo-EM data collection. A.C.M.C. contributed to developing the purification protocol and assisted with model building. A.S. and H.U. carried out mass spectrometry analysis. P.C. supervised research. H.W. and P.C. interpreted the data and wrote the manuscript, with input from all authors.

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.

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 Cryo-EM structure determination and analysis of SAGA.

Related to data shown in Fig. 1. a, Purification of endogenous SAGA from S. cerevisiae. SDS–PAGE of peak fraction used for cryo-EM grid preparation. Identity of the bands was confirmed by mass spectrometry. For gel source data, see Supplementary Fig. 1. b, Exemplary cryo-EM micrograph of data collection. The micrograph is shown before (left) and after (right) denoising using Warp35. c, The 2D class averages. d, Sorting and classification tree used to reconstruct SAGA. e, FSC between half maps of the final reconstructions of the complete SAGA complex and the SAGA modules Tra1 and core. Resolutions for the gold-standard FSC 0.143 criterion are listed. f, Angular distribution plot for all particles in the final reconstructions of the SAGA core (top) and Tra1 (bottom) modules. Colour shading from blue to yellow correlates with the number of particles at a specific orientation as indicated.

Extended Data Fig. 2 Quality of the SAGA structure.

Relates to data in Figs. 1, 2. a, SAGA reconstruction coloured according to local resolution43. Model–map FSC curves calculated between the refined atomic models and maps are shown below. b, Electron density (grey transparent surface) for various SAGA regions as indicated. c, Overview of the cross-linking data. Circular plot of high-confidence lysine–lysine intersubunit (green) and intrasubunit (purple) cross-links obtained by mass spectrometry for the SAGA complex. The mass spectrometry measurement was repeated twice independently with similar results. Totals of 396 unique intersubunit cross-links and 514 intrasubunit cross-links were obtained. d, Validated cross-links mapped onto the SAGA structure. Out of 396 unique intersubunit cross-links, 120 could be mapped onto the core-module structure, and 109 were located within the 30 Å distance limit for the BS3 cross-linker. Blue lines depict the cross-links with cross-linked sites within the 30 Å distance permitted by BS3, whereas red lines depict cross-links over more than 30 Å.

Extended Data Fig. 3 Comparison of the histone-like fold in SAGA with the histone octamer, details of Taf5–Spt20 interactions, and model of the SAGA–TBP complex.

Relates to data in Figs. 13. a, Comparison of the SAGA core module histone octamer-like structure with the canonical histone octamer core (PDB: 1AOI). The canonical octamer core is rendered as the colour for the SAGA octamer-like fold. b, Details of Taf5–Spt20 wedge interactions. Residues involved in the interactions are shown in sticks and coloured as indicated. c, Details of interactions between the Taf5 LisH domain and Spt20 SEP domain. Residues involved in the interactions are shown in sticks and coloured as indicated. d, Model of the SAGA–TBP complex. The model was generated by superposing the TBP-containing TFIID lobe A onto the SAGA core structure. A homology model for Spt8 was generated by the I-TASSER server44.

Extended Data Fig. 4 Details of intermodule interactions.

Relates to data in Figs. 1, 2. a, Binding interface between core and Tra1 modules. The Tra1 FAT domain (grey) is shown as a surface representation. The TIRs of Taf12 (green) and Spt20 (yellow) are shown in cartoon representation. b, Details of the interactions depicted in a. c, Sgf73 (turquoise) tethers the DUB module to the core module. Residues involved in the interactions are shown in sticks and coloured as indicated. d, Sequence alignment of SAGA subunit regions involved in intermodule interactions. Conserved residues are highlighted in blue. Key residues are labelled with asterisks45. Sc, S. cerevisiae; Pp, Pichia pastoris; Sp, Schizosaccharomyces pombe.

Extended Data Fig. 5 Cryo-EM structure determination and analysis of the SAGA–nucleosome complex.

Relates to data in Fig. 4. a, Exemplary cryo-EM micrograph of data collection. The micrograph is shown before (left) and after (right) denoising using Warp35. b, The 2D class averages for the SAGA–nucleosome complex. c, The 2D class averages for the DUB module–nucleosome subcomplex. d, Sorting and classification tree used to reconstruct the DUB module–nucleosome complex at 3.7 Å resolution. e, FSC between half maps of the final reconstructions of the SAGA module, Tra1 and the DUB module–nucleosome complex from SAGA–nucleosome complex data. Resolutions for the gold-standard FSC 0.143 criterion are listed. f, Angular distribution plot for all particles in the final reconstruction of the SAGA DUB module–nucleosome complex. Colour shading from blue to yellow correlates with the number of particles at a specific orientation as indicated. g, Superposition of the crystal structure of DUB-ubiquitinated nucleosome (4ZUX)12 onto the cryo-EM structure presented here. Structures are shown in cartoon and coloured as indicated. h, Comparison of the low-pass-filtered overall cryo-EM maps of SAGA and the SAGA–nucleosome complex. Densities for the HAT and DUB modules are lost on nucleosome binding to SAGA.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
Extended Data Table 2 Modelling of yeast SAGA subunits, domains and regions
Extended Data Table 3 Conservation of SAGA between yeast and human

Supplementary information

Supplementary Figures

Supplementary Figure 1: This file contains an uncropped scan with size marker indication (cf. Extended Data Fig. 1a).

Reporting Summary

Supplementary Table

List of BS3 crosslinks within SAGA: List of intra- and inter-subunit lysine-lysine crosslinks as identified by LC-MS analyses and subsequent database search using pLink 138 and pLink 239. The respective scores of cross-link identification are listed as well as the number of CSMs (cross-linked spectra matches).

Video 1

Overview of SAGA structure The video shows a vertical rotation of the SAGA complex structure. It first depicts the overall shape of SAGA based on a low pass-filtered cryo-EM density map. It then shows the high-resolution cryo-EM reconstructions for the Tra1 and core modules, respectively. Finally, it shows the Tra1, core, and DUB module structures fitted to the densities.

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Wang, H., Dienemann, C., Stützer, A. et al. Structure of the transcription coactivator SAGA. Nature 577, 717–720 (2020). https://doi.org/10.1038/s41586-020-1933-5

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