Nature | Vol 577 | 30 January 2020 | 711Article
Structure of SAGA and mechanism of TBP
deposition on gene promoters
Gabor Papai1,2,3,4, Alexandre Frechard1,2,3,4, Olga Kolesnikova1,2,3,4, Corinne Crucifix1,2,3,4,
Patrick Schultz1,2,3,4* & Adam Ben-Shem1,2,3,4*SAGA (Spt–Ada–Gcn5–acetyltransferase) is a 19-subunit complex that stimulates
transcription via two chromatin-modifying enzymatic modules and by delivering the
TATA box binding protein (TBP) to nucleate the pre-initiation complex on DNA, a
pivotal event in the expression of protein-encoding genes^1. Here we present the
structure of yeast SAGA with bound TBP. The core of the complex is resolved at 3.5 Å
resolution (0.143 Fourier shell correlation). The structure reveals the intricate
network of interactions that coordinate the different functional domains of SAGA and
resolves an octamer of histone-fold domains at the core of SAGA. This deformed
octamer deviates considerably from the symmetrical analogue in the nucleosome and
is precisely tuned to establish a peripheral site for TBP, where steric hindrance
represses binding of spurious DNA. Complementary biochemical analysis points to a
mechanism for TBP delivery and release from SAGA that requires transcription factor
IIA and whose efficiency correlates with the affinity of DNA to TBP. We provide the
foundations for understanding the specific delivery of TBP to gene promoters and the
multiple roles of SAGA in regulating gene expression.Transcription of protein-encoding genes begins with the formation of a
pre-initiation complex (PIC) comprising RNA polymerase II and several
general transcription factors^2. PIC assembly is nucleated by loading TBP
onto promoter DNA^3 , a focal point for regulated gene expression^4. Two
multiprotein complexes, transcription factor IID (TFIID) and SAGA,
can deliver TBP to gene promoters^5 ,^6 and are required for global gene
expression in yeast^7 ,^8.
The 1.6-MDa SAGA complex also stimulates transcription via its two
chromatin-modifying enzymatic activities. The complex comprises
19 subunits organized in four modules with distinct functions^2 ,^9 : a his-
tone acetyltransferase (HAT) module, a histone deubiquitinase (DUB)
module, the 430-kDa Tra1 subunit that serves as a docking platform for
transcription factors that recruit SAGA to activating DNA sequences
upstream of the promoter, and a 10-subunit central module that is
physically connected to all other modules and is responsible for recruit-
ing TBP to SAGA (Extended Data Fig. 1a).
Structures of several individual isolated components of SAGA, nota-
bly the DUB module^10 ,^11 , the catalytic subunit of the HAT module (Gcn5)^12
and Tra1^13 ,^14 have been obtained. Previous electron microscopy studies
positioned Tra1 in one lobe of the SAGA complex, but all other subunits
appeared together as a main lobe that could only be described at low
resolution^15 ,^16.
The central module of yeast SAGA shares five TBP-associated factors
(TAFs) with TFIID. Both complexes also contain multiple histone-fold
domains that associate into heterodimers and higher-order oligomers^17 ,^18
(Extended Data Fig. 1a, b). The central module is therefore expected to
be structurally related to TFIID, suggesting that TBP binding in both
complexes shares some common features. Progress in defining the
architecture of TFIID was reported recently; however, the machinery
responsible for TBP binding and delivery remained poorly resolved^19 ,^20.
Here we report the 3.5 Å-resolution structure of the full SAGA com-
plex bound to TBP, determined from cryo-electron microscopy (cryo-
EM) maps that show clear density for the majority of the protein side
chains. Our structure describes in atomic detail the network of inter-
actions that coordinate the functional domains of SAGA as well as a
machinery precisely geared to binding TBP and preventing aberrant
transcription initiation. With complimentary experimental findings,
our structure points to a mechanism of TBP delivery and release from
SAGA that depends on TFIIA and the affinity of promoter DNA to TBP,
providing new insights into regulation of PIC assembly.Overall architecture of SAGA
We reconstituted the complex with SAGA purified from the yeast
Komagataella phaffii (also known as Pichia pastoris), full length TBP
and ubiquitin aldehyde and collected a dataset of more than one mil-
lion particles. Image analysis resulted in a map of the full SAGA com-
plex at 3.8 Å resolution, calculated from 338,850 selected particles
(Fig. 1a, Extended Data Figs. 1c, d, 2, Supplementary Video 1, Extended
Data Table 1). Focused refinement of the main lobe and of Tra1 further
improved their average resolution to 3.5 Å, with many regions reaching
as high as 3 Å. Hence, Tra1 and the central module, as well as the domains
of Sgf73 that anchor the DUB to the central module, show clear densi-
ties for the majority of side chains (Fig. 1b, Extended Data Fig. 2b, c).
At the periphery of SAGA, exposed next to Spt3, we identified the
distinctive saddle-shaped density of the conserved core of TBP (cTBP),https://doi.org/10.1038/s41586-020-1944-2
Received: 20 June 2019
Accepted: 19 November 2019
Published online: 22 January 2020
(^1) Institut de Génétique et de Biologie Moléculaire et Cellulaire, Integrated Structural Biology Department, Equipe labellisée Ligue Contre le Cancer, Illkirch, France. (^2) Centre National de la
Recherche Scientifique, UMR7104, Illkirch, France.^3 Institut National de la Santé et de la Recherche Médicale, U1258, Illkirch, France.^4 Université de Strasbourg, Illkirch, France. *e-mail: patrick.
[email protected]; [email protected]