Structure of Transcription Coactivator SAGA

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Structure of Transcription Coactivator SAGA Wang et al.: Structure of transcription coactivator SAGA 1 Structure of transcription coactivator SAGA 2 3 Haibo Wang1, Christian Dienemann1, Alexandra Stützer2, Henning Urlaub2,3, Alan C.M. 4 Cheung4,5, and Patrick Cramer1 5 6 1Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, 7 Göttingen, Germany. 2Max Planck Institute for Biophysical Chemistry, Bioanalytical Mass 8 Spectrometry, Göttingen, Germany. 3University Medical Center Göttingen, Institute of 9 Clinical Chemistry, Bioanalytics Group, Göttingen, Germany. 4Department of Structural 10 and Molecular Biology, Institute of Structural and Molecular Biology, University College 11 London, London, United Kingdom. 5Institute of Structural and Molecular Biology, Biological 12 Sciences, Birkbeck College, London, United Kingdom. 13 14 Gene transcription by RNA polymerase II is regulated by activator proteins that 15 recruit the coactivator complexes SAGA1,2 and TFIID2-4. SAGA is globally required 16 for regulated transcription5 and conserved amongst eukaryotes6. SAGA contains four 17 modules7-9, the activator-binding Tra1 module, the core module, the histone 18 acetyltransferase (HAT) module, and the histone deubiquitination (DUB) module. 19 Previous works provided partial structures10-14, but the structure of the central core 20 module is unknown. Here we present the cryo-electron microscopy structure of SAGA 21 from the yeast Saccharomyces cerevisiae and resolve the core module at 3.3 Å 22 resolution. The core module consists of subunits Taf5, Sgf73 and Spt20, and a histone 23 octamer-like fold. The octamer-like fold comprises the heterodimers Taf6-Taf9, 24 Taf10-Spt7 and Taf12-Ada1, and two histone-fold domains in Spt3. Spt3 and the 25 adjacent subunit Spt8 interact with the TATA box-binding protein (TBP)2,7,15-17. The 26 octamer-like fold and its TBP-interacting region are similar in TFIID, whereas Taf5 27 and the Taf6 HEAT domain adopt distinct conformations. Taf12 and Spt20 form 28 flexible connections to the Tra1 module, whereas Sgf73 tethers the DUB module. 29 Binding of a nucleosome to SAGA displaces the HAT and DUB modules from the core 30 module surface, allowing the DUB module to bind one face of an ubiquitinated 31 nucleosome. 32 33 SAGA contains 19 subunits that are distributed over four modules18. The Tra1 module binds 34 activators19,20, the core module recruits TBP21, the HAT module contains the histone H3 35 acetyltransferase Gcn51, and the DUB module comprises a histone H2B deubiquitinase22,23. 36 To determine the structure of SAGA, we purified the endogenous complex from 37 Saccharomyces cerevisiae using a strain with a C-terminal TAP-tag on subunit Spt20 38 (Methods). Purified SAGA contained all 19 subunits in apparently stoichiometric amounts 39 (Extended Data Fig. 1a), and was subjected to cryo-electron microscopy (cryo-EM) and 40 protein crosslinking analysis (Methods). We obtained a reconstruction of SAGA at an 41 overall resolution of 3.9 Å (Extended Data Figure 1; Supplementary Video 1). 1 Wang et al.: Structure of transcription coactivator SAGA 42 The two large SAGA modules, the Tra1 and the core module, were resolved at 3.4 Å 43 and 3.3 Å resolution, respectively (Extended Data Figures 1d-e, 2a). We fitted the Tra1 44 structure13, built the core module and the protein regions connecting the two modules, and 45 refined the structure in real space (Extended Data Tables 1, 2; Extended Data Figure 2b). 46 The HAT and DUB modules were more flexible and showed resolutions of 9 Å and 12 Å, 47 respectively. The structure of the DUB module12 could be fitted, but density for the HAT 48 module could not be interpreted (Supplementary Video 1). Our protein-protein 49 crosslinking and published crosslinking information7 confirmed our modelling and 50 assigned subunit Spt8 to a remaining density located between the core and Tra1 modules 51 (Supplementary Table 1; Extended Data Figure 2c, d). 52 The structure confirms the overall topology of SAGA with four flexibly connected 53 modules8,24, and reveals the intricate subunit architecture of the coactivator complex 54 (Fig. 1). The SAGA structure contains only one copy of each subunit, in contrast to TFIID, 55 which contains two copies of several subunits3,4. The SAGA core module occupies a central 56 position and comprises the subunits Taf5, Taf6, Taf9, Taf10, Taf12, Spt3, Spt7, Spt20, and 57 Ada1. The TBP-interacting subunit Spt8 is flexibly connected to the core module, as are 58 the HAT and DUB modules (Fig. 1a). These three functional SAGA regions are lined up 59 on one side of the complex that is predicted to face promoter DNA (Fig. 1a). 60 The core module contains a histone octamer-like fold and an adjacent submodule 61 formed by subunits Taf5, Taf6 and Spt20 (Fig. 2). The octamer-like fold comprises three 62 pairs of subunits that each contribute one histone fold (HF), namely Taf6-Taf9, Taf10-Spt7 63 and Taf12-Ada1, and by Spt3, which contributes two HFs. The presence of an octamer-like 64 fold explains early observations of histone-like subunit pairs in SAGA25,26. In contrast to a 65 canonical histone octamer, which shows two-fold symmetry, the SAGA octamer-like fold 66 is fully asymmetric (Extended Data Fig. 3a). 67 Subunit Taf5 connects the octamer-like fold to the remainder of the core module 68 and is thus important for core module architecture (Fig. 2b). The N-terminal helical domain 69 of Taf5 binds the C-terminal HEAT repeat region of Taf6. The C-terminal WD40 b- 70 propeller of Taf5 docks to the HF pairs Taf6-Taf9 and Taf10-Spt7, and binds Spt20, which 71 contains a SEP (shp1-eyc-p47) domain (Fig. 2c). Spt20 also contains an extended loop that 72 forms a wedge between the two Taf5 domains, thereby stabilizing them in a defined 73 orientation (Fig. 2b, Extended Data Fig. 3b). The Lis1 homology motif (LisH) helices of 74 Taf5 interact with the SEP domain of Spt20 (Extended Data Fig. 3c). Taf6 contributes one 75 b-strand to the Taf5 propeller, suggesting that Taf5 and Taf6 form an obligate heterodimer 76 (Fig. 2c). The Taf5 propeller and the Taf6 HEAT region both interact with a stretch of Spt7 77 that extends from the octamer-like fold and continues into a bromodomain that is mobile. 78 SAGA has long been known to bind TBP15-17,27, implying a role in recruiting TBP 79 to promoters. According to crosslinking7 and genetic data2, SAGA interacts with TBP using 80 its subunits Spt3 and Spt8. These two subunits occupy adjacent locations at the edge of the 81 octamer-like fold (Fig. 1, Fig. 3a). SAGA and TFIID share the subunits Taf5, Taf6, Taf9, 2 Wang et al.: Structure of transcription coactivator SAGA 82 Taf10, and Taf1218. TFIID consists of three lobes that were called lobe A, B, and C and 83 were structurally defined recently3,4. Lobe A contains an octamer-like fold that resembles 84 the fold observed in SAGA. However, Spt7 and Ada1 are replaced by TFIID subunits Taf3 85 and Taf4, respectively, and the two HF domains of Spt3 are replaced by the HF pair Taf11- 86 Taf13 in TFIID (Fig. 3b). Despite these differences, the octamer-like folds in SAGA and 87 TFIID bind TBP at the same relative position (Fig. 3a, b). 88 We generated a model of the SAGA-TBP complex by superposing the TBP- 89 containing TFIID structure lobe A onto the SAGA core structure (Extended Data Fig. 3d). 90 The model is consistent with TBP bridging between SAGA subunits Spt3 and Spt8. In 91 TFIID, the TFIID-specific subunit Taf1 also contributes to TBP binding, and this may 92 explain an apparently higher affinity of TFIID for TBP compared to SAGA2. TFIID lobe B 93 contains a hexamer of HF domains that lacks the Taf11-Taf13 pair and does not bind TBP, 94 but otherwise resembles its counterpart in SAGA (Fig. 3c). 95 Structural comparisons also show how SAGA and TFIID form distinct structures 96 despite sharing five subunits (Fig. 3). The shared subunits Taf5 and Taf6 have diFferent 97 structures in the two complexes. In TFIID, the Taf5 N-terminal domain docks to the 98 octamer-like fold and is stabilized by the TFIID-specific subunit Taf4. In SAGA, Taf4 is 99 absent and subunit Ada1 occupies its position. The Taf5 N-terminal domain occupies a 100 position that is distant from the octamer-like fold, is stabilized by the SAGA-specific 101 subunit Spt20, and contacts the Taf6 HEAT repeat domain, which also adopts a diFferent 102 position and structure compared to TFIID. Thus, incorporation of Ada1 or Taf4 into the 103 octamer-like fold may trigger assembly of either SAGA or TFIID, respectively. 104 Our results also show that the core module forms flexible connections to the other 105 modules of SAGA. The core subunits Taf12 and Spt20 contain Tra1-interacting regions 106 (TIRs) that tether the Tra1 module. The Taf12 TIR (residues 353-410) meanders through a 107 narrow surface groove formed by the TPR repeats of the FAT domain in Tra1, whereas 108 Spt20 contains two TIRs (Extended Data Figure 4a, b, d). TIR1 (residues 398-416) forms 109 a latch that retains the Taf12 TIR, and TIR2 (residue 474-488) forms a helix that binds Tra1 110 (Extended Data Figure 4a, b, d). These interactions are consistent with the known 111 dissociation of Tra1 upon Spt20 deletion28. Further, subunit Sgf73 connects the core to the 112 DUB module (Extended data Figure 4c, d). Whereas the central Sgf73 residues 353-437 113 are part of the core module (‘anchor helices’) and form interactions with Spt20, Ada1 Taf12, 114 Taf6 and Taf9, the ~100 N-terminal residues are part of the DUB module10-12.
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