Human TFIID Binds
to Core Promoter DNA
in a Reorganized Structural State
Michael A. Cianfrocco,1George A. Kassavetis,4Patricia Grob,2Jie Fang,2Tamar Juven-Gershon,5James T. Kadonaga,4
and Eva Nogales2,3,6,*
1Biophysics Graduate Group
2Howard Hughes Medical Institute
3Department of Molecular and Cell Biology
University of California, Berkeley, Berkeley, CA 94720, USA
4Division of Biological Sciences, Section of Molecular Biology, University of California, San Diego, La Jolla, CA 92093-0347, USA
5Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel
6Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
A mechanistic description of metazoan transcription
is essential for understanding the molecular pro-
cesses that govern cellular decisions. To provide
structural insights into the DNA recognition step of
transcription initiation, we used single-particle elec-
tron microscopy (EM) to visualize human TFIID with
promoter DNA. This analysis revealed that TFIID
coexists in two predominant and distinct structural
states that differ by a 100 A˚translocation of TFIID’s
is modulated by TFIIA, as the presence of TFIIA and
promoter DNA facilitates the formation of a rear-
ranged state of TFIID that enables promoter recogni-
tion and binding. DNA labeling and footprinting,
together with cryo-EM studies, were used to map
(MTE), and downstream core promoter element
(DPE) promoter motifs within the TFIID-TFIIA-DNA
structure. The existence of two structurally and func-
tionally distinct forms of TFIID suggests that the
different conformers may serve as specific targets
for the action of regulatory factors.
The regulation of gene expression is a complex task that is crit-
ical for the growth, development, and survival of organisms.
Underlying its importance is the fact that, although the number
of protein coding genes has remained fairly constant throughout
metazoan evolution, the number of regulatory DNA elements has
increased dramatically (Levine and Tjian, 2003). By fine-tuning
both the rate and synchrony of transcription initiation by RNA
polymerase II (RNAPII), transcriptional regulation can serve as
a key control point to produce organism-wide changes in gene
expression profiles in response to developmental and environ-
mental cues (Levine, 2011).
The initiation of transcription by RNAPII requires basal tran-
scription factors known as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and
TFIIH (Thomas and Chiang, 2006). These factors assemble
onto the core promoters of protein coding genes to form a tran-
scription preinitiation complex (Buratowski et al., 1989; Rhee
and Pugh, 2012). A sequential recruitment model has been
proposed whereby TFIID and Mediator serve as coactivators
that facilitate interactions between upstream and promoter
proximal factors (Burley and Roeder, 1996). The interaction of
TFIID with promoter DNA can be further stabilized through a
TFIIA-mediated release of the inhibitory N-terminal domain of
TAF1 from the concave DNA-binding surface of TATA-binding
protein (TBP) (Bagby et al., 2000; Geiger et al., 1996; Liu et al.,
1998). The formation of the TFIID-TFIIA-DNA complex is then
followed by the binding of TFIIB, RNAPII, TFIIF, TFIIE, and TFIIH
to yield the transcriptionally competent preinitiation complex
(Thomas and Chiang, 2006).
TFIID is a multisubunit complex that comprises TBP and ?12–
13 proteins termed TBP-associated factors (TAFs) (Burley and
Roeder, 1996). Through the use of TBP and TAFs, TFIID makes
sequence-specific contacts with core promoter DNA elements,
including the TATA box, Initiator (Inr), motif ten element (MTE),
downstream core promoter element (DPE), and downstream
core element motifs (Juven-Gershon and Kadonaga, 2010).
These interactions have been exploited to create a highly active
core promoter, termed the super core promoter (SCP), that
is capable of engaging in high-affinity interactions with TFIID
through the presence of optimal versions of the TATA, Inr,
MTE, and DPE motifs (Juven-Gershon et al., 2006).
Despite the importance of TFIID as a coordinator of transcrip-
tion initiation, high-resolution structural information has been
restricted to crystal structures of a small number of subunits
and domains within TFIID (Bhattacharya et al., 2007; Jacobson
et al., 2000; Kim et al., 1993a, 1993b; Liu et al., 1998; Werten
etal.,2002;Xie etal.,1996).Thesizeand scarcityofTFIID,which
typically is purified from endogenous sources, has restricted
120 Cell 152, 120–131, January 17, 2013 ª2013 Elsevier Inc.
structural studies to methods that require microgram quantities
of sample. Single-particle electron microscopy (EM) has proven
to be an indispensable tool for the structural characterization of
largemultisubunit complexes, evenwhenthe sample isavailable
in minute amounts. This technique also has the potential to
characterize the structural dynamics of large protein complexes
(Leschziner and Nogales, 2007).
EM structural studies of TFIID have yielded low-resolution
structures (20–30 A˚) of yeast and human TFIID (Andel et al.,
1999; Brand et al., 1999; Elmlund et al., 2009; Grob et al., 2006;
Leurent et al., 2002, 2004; Liu et al., 2009; Papai et al., 2009,
2010). A number of these studies suggested a role of conforma-
tional flexibility in promoter binding by TFIID. Recently, several
groups have reported single-particle EM structures of purified
endogenous yeast TFIID bound to promoter DNA (Elmlund
et al., 2009; Papai et al., 2010). These studies examined the
romyces cerevisiae to promoters that contain both TATA and
Inr sequence elements. They used computational methods to
sort TFIID into distinct conformational states and localized addi-
tional densities, which were attributed to TATA box DNA, to the
surfaces of their structures.
To gain insight into the function of metazoan TFIID, we applied
single-particle EM and extensive image-sorting methods to
characterize the structural dynamics of human TFIID. We found
that TFIID exhibits a surprising degree of flexibility, moving its
lobe A (?300 kDa in size) by 100 A˚across the central channel
of TFIID in a dynamic equilibrium. These findings reveal a rear-
ranged conformation of TFIID that corresponds to a high-affinity
DNA-binding state. By using gold labeling and multimodel
refinements, we were able to define the organization of the
rearranged conformation of TFIID bound to promoter DNA. We
probed the protein–DNA interactions further by carrying out
DNA footprinting experiments with DNase I and methidium-
propyl-EDTA-Fe(II), and mapped the digestion patterns on
wild-type and mutant SCP onto the cryo-EM structure of
TFIID-TFIIA-SCP. These studies suggest a model in which the
distinct conformations of TFIID may serve as targets through
which regulatory factors recruit TFIID to specific types of core
Lobe A Is Flexibly Attached to a Stable Core of TFIID that
Comprises Lobes B and C
In a previous cryo-EM analysis of human TFIID, the use of a
three-dimensional (3D) variance approach indicated that confor-
mational flexibility is an intrinsic property of TFIID (Grob et al.,
2006). Given the potential role of this flexibility in TFIID function,
we investigated this property more thoroughly by performing an
extensive two-dimensional (2D) image analysis of negatively
stained TFIID samples. When we concentrated specifically on
a standard view in which the three main lobes of TFIID (lobes
A, B, and C) were clearly separated and distinguishable, we
were surprised to find that lobe A was present in a range of posi-
tions with respect to a more stable core of the complex formed
by lobes B and C (termed the BC core) (Figure S1A available
reorganization of TFIID that was not fully appreciated in previous
work on human TFIID.
We further examined these substantial differences in the posi-
tioning of lobe A within TFIID. Subclassification within a 2D mask
that excluded the stable BC core revealed that lobe A adopts
a wide range of positions (Figures S1B and S1C). This analysis
recovered a previously described structure in which lobe A is
located near lobe C, and we will refer to this arrangement as
the canonical state of human TFIID (Andel et al., 1999; Grob
et al., 2006; Liu et al., 2008, 2009). In addition, we found that
lobe A can also be positioned across the TFIID central channel
at a location in which it contacts lobe B (Figure S1C). In this
conformation, TFIID forms a rearranged, horseshoe-like struc-
ture wherein the order of the lobes has been altered. We refer
to this conformation as the rearranged state (Figure S1C). To
rule out the possibility that these distinct positions of lobe A
were an artifact due to the negative-staining procedures, we
carried out an identical analysis using cryo-EM images of the
same TFIID sample (Figure S1D). A similar range of positions of
lobe A with respect to a stable BC core was detected in the
frozen-hydrated TFIID sample, thus confirming the coexistence
of these distinct states of the complex (Figure 1; Movie S1).
To quantify the structural plasticity exhibited by TFIID, we
measured the position of lobe A along the BC core within
each subclassified average. The measurements were normal-
ized such that values >0.70 correspond to particles resembling
the canonical state, in which lobe A is at its closest position to
lobe C (Figure S1E). Values <0.50, on the other hand, indicate
that the particles are in a rearranged state, wherein lobe A is
proximal to lobe B (Figure S1E). This analysis revealed that the
position of lobe A can be described by a bimodal distribution
with peaks centered at 0.40 and 0.80 (Figure 2A). Surprisingly,
this analysis showed that ?50% of the TFIID particles are found
in the rearranged state. These distinct structural states of TFIID
Figure 1. Lobe A Exists in a Range of Positions Relative to a Stable
BC Core within the Purified TFIID Complex
2D reference-free cryo-EM class averages of TFIID are shown alongside
contour models to indicate lobe positions within each average. Lobe A is
colored yellow and the stable BC core is colored blue. Scale bar in (A) is 200 A˚.
See also Figure S1 and Movie S1.
Cell 152, 120–131, January 17, 2013 ª2013 Elsevier Inc. 121
were observed in data sets from both cryo-EM and negative-
stain data, demonstrating that the sample preparation did not
alter the results (Figure S1F). Hence, there are two predominant
and structurally distinct states of TFIID that differ with regard to
the reorganization of lobe A within the complex.
The Conformational Landscape of TFIID Is Affected by
TFIIA and Promoter DNA
To examine whether the binding of TFIIA and promoter DNA is
linked to the conformational states of TFIID, we analyzed TFIID
in the presence of excess SCP DNA and/or the cofactor TFIIA.
The SCP contains the TATA box, Inr, MTE, and DPE core
Figure 2. TFIID’s Conformational Land-
scape Changes in Response to TFIIA and
(A–D) Distribution of lobe A positions relative to the
stable BC core for TFIID (A), TFIID-SCP (B), TFIID-
TFIIA (C), and TFIID-TFIIA-SCP samples (D). Inset
in (A): Class averages corresponding to specific
lobe A measurements from the TFIID sample.
See also Figure S1.
promoter motifs. When TFIID was incu-
bated with SCP DNA, and negatively
stained samples were visualized and
analyzed as described above, lobe A
showed a similar distribution of posi-
tions relative to TFIID (p = 0.0353,
Wilcoxon signed-rank test; Figure 2B).
This result suggested either that TFIID
does not interact with SCP under the
conditions used, or that DNA binding
does not alter the conformational parti-
tioning of lobe A within the accuracy of
TFIIA affects the properties of TFIID.
When TFIID was incubated with TFIIA in
the absence of DNA, the distribution of
lobe A positions was shifted to the peak
centered at 0.80. It thus appears that
TFIIA stabilizes TFIID in the canonical
state (Figure 2C). On the other hand,
in the presence of SCP DNA, TFIIA pro-
motes the formation of the rearranged
state of TFIID, with a strong increase in
the peak of lobe A positions centered
at 0.40 (Figure 2D). The distribution of
lobe A positions within the context of
TFIID-TFIIA (Figure 2C) was significantly
different from that seen with TFIID-TFIIA-
SCP (p = 1.5 3 10?13, Wilcoxon signed-
rank test; Figure 2D). Because TFIID was
purified according to established proto-
constant between these experiments,
suggesting that these conformational changes are due to the
presence of TFIIA and SCP DNA. These results indicate that
TFIIA serves the dual function of maintaining TFIID in the canon-
ical state in the absence of DNA and promoting the formation
of the rearranged state in the presence of promoter DNA.
The Rearranged State of TFIID Binds SCP DNA in the
Presence of TFIIA
To characterize the structure of this rearranged state of TFIID
and its binding to DNA, we carried out cryo-EM visualization
of frozen hydrated samples. An analysis of 2D reference-free
averages from cryo-EM data of the TFIID-TFIIA-SCP ternary
122 Cell 152, 120–131, January 17, 2013 ª2013 Elsevier Inc.
complex showed the presence of both the rearranged and
canonical states (Figures 3A and 3B, leftmost panels). Impor-
tantly, the class averages corresponding to the rearranged state
revealed extra density that appeared to be DNA extending
across the central channel of TFIID (Figure 3A, left panel). We
confirmed that this additional density was DNA by examining
cryo-EM 2D reference-free class averages collected for TFIID,
TFIID-SCP, and TFIID-TFIIA. The 2D reference-free class aver-
ages of TFIID without the combined presence of TFIIA and
SCP showed no extra density within the rearranged state (Fig-
ure 3A). These observations suggest that TFIIA is required for
efficient binding of TFIID to SCP, and that the predominant
form of TFIID that is bound to DNA is the rearranged state.
Cryo-EM Structures of the Rearranged and Canonical
States of TFIID
The 2D image analysis indicated that both TFIID and the ternary
TFIID-TFIIA-SCP samples exist in a distribution of conforma-
tional states (Figures 2A and 2D); therefore, cryo-EM recon-
structions of these samples needed to involve a multimodel 3D
refinement strategy. We sorted a total of ?35,000 cryo-EM
particle images from the purified TFIID-TFIIA-SCP complex
using multireference projection matching with two distinct ab
initio references structures that were obtained using the orthog-
onal tilt reconstruction method (Leschziner and Nogales, 2006;
Figure S2; Extended Experimental Procedures) and represent
the rearranged and canonical conformations. After implement-
ing a cross-correlation cutoff that excluded 25% of the particles,
we refined the reconstruction of the rearranged state (com-
prising ?60% of the remaining particles) to a resolution of 32 A˚
(Figures 3C and S3B–S3D). A prominent feature of the rear-
ranged structure is the presence of density (Figure 3C, green),
which we attribute to DNA, extending over lobe C and across
the central channel of TFIID toward the region connecting lobes
A and B. Although the DNA density exhibits one location of weak
Figure 3. TFIIA-Mediated Binding of SCP DNA to the Rearranged State of TFIID
(A and B) Cryo-EM 2D reference-free class averages of the rearranged conformation (A) and the canonical conformation (B) shown alongside contour models.
(Cand D) 3Dreconstructionsof therearrangedconformationfor TFIID-TFIIA-SCPat32A˚(C) and TFIIDat 35A˚(D).LobeA is inyellowand the densityattributedto
DNA in (C) is shown in green.
and lobe A is in orange. Scale bar is 200 A˚.
See also Figures S2, S3, and S7.
Cell 152, 120–131, January 17, 2013 ª2013 Elsevier Inc. 123
TFIID-TFIIA-SCP (?1.2 MDa), a continuous density can be ob-
served across the central cavity of TFIID at a slightly lower
threshold (Figure S3A).
A 3D model for the canonical state was refined simultaneously
from the TFIID-TFIIA-SCP samples to a similar resolution and
included the remaining 40% of the selected particles (Figures
3E and S3B–S3D). Importantly, the canonical state does not
show the presence of any apparent DNA density, but is other-
wise similar in overall features to the previously reported cryo-
EM (Grob et al., 2006) and negative-stain (Liu et al., 2009)
structures of human TFIID. A comparison of the coexisting
canonical and rearranged structures confirms the presence of
tion of lobe A is dramatically shifted from one side of the core
to the other, where lobe A is within close proximity of the DNA
binding site of lobe C (Figure 3C, yellow, and Figure 3E, orange).
The movement of lobe A from the canonical state to the
rearranged state involves a translational component along the
BC core axis (Movie S2), aslobe Achanges its apparent connec-
tivity from lobe C (canonical) to lobe B (rearranged). These two
Figure 4. Organization of Promoter DNA
and TFIIA withinthe
(A) Cryo-EM reconstruction
SCP(?66) at 35 A˚
the cryo-EM reconstruction of TFIID-TFIIA-SCP
(center) and their difference map (right). DNA
density is colored in green, and lobe A and the BC
core are colored in yellow and blue, respectively.
Scale bar is 100 A˚.
(B–D) 2D reference-free class averages for the
rearranged TFIID-TFIIA-SCP complex with Nano-
gold labels on SCP DNA at +45 (B), SCP DNA at
TATA (C), and TFIIA (D).
(E) 2D reference-free class average for the
canonical state of TFIID-TFIIA-SCP containing
Nanogold labeled on TFIIA. 3D models are
shown alongside high-defocus averages (density
threshold at s = 3.5) with a gold sphere marking
the localization of Nanogold for each experiment.
See Extended Experimental Procedures for a
detailed discussion about generating low-defocus
class averages. Summary of gold labeling results
for (B)–(D) are shown in (A), second row.
(left column) aligned with
reconstructions from the TFIID-TFIIA-
SCP sample suggest that TFIID ex-
hibits high-affinity DNA interactions only
when it adopts the rearranged con-
formation, considering the lack of any
apparent DNA density for the canonical
The assignment of DNA as the narrow,
linear density present only in the rear-
ranged state of the TFIID-TFIIA-SCP
sample was further supported by a
comparison of the cryo-EM structure of
the rearranged state with that of the
approach described above, we obtained 3D cryo-EM recon-
structions of the two alternative states of TFIID (Figures 3D, 3F,
and S3F–S3H). A comparison of the rearranged conformation
(?50% of selected particles) of TFIID with the structure of
TFIID-TFIIA-SCP shows that there is strong density, which we
ascribe to DNA, that is uniquely in the ternary complex and
extends across the TFIID channel from lobe C to lobe A (Figures
3C, 3D, S3A, and S3E). The structures of the canonical confor-
mation of TFIID observed with TFIID alone and with the TFIID-
TFIIA-SCP sample appear to be similar (Figures 3E and 3F;
Movie S2), which suggests that the particles in the canonical
conformation in the TFIID-TFIIA-SCP sample lack stably posi-
tioned SCP DNA.
Promoter DNA Path through TFIID
We then examined the orientation of the DNA bound to the
rearranged TFIID-TFIIA-SCP complex. First, we collected cryo-
EM data from a sample of TFIID, TFIIA, and SCP DNA with
a 30 bp 50extension to position ?66 (termed ?66) upstream
of the TATA box (Figure 4A). A comparison of cryo-EM 3D
124 Cell 152, 120–131, January 17, 2013 ª2013 Elsevier Inc.
reconstructions for the rearranged state of TFIID-TFIIA-
SCP(?66) and TFIID-TFIIA-SCP revealed significant extra den-
sity extending out of lobe A, which was the strongest difference
between the two cryo-EM reconstructions at s = 4 (Figure 4A,
arrowheads; Movie S2). The dimensions of this additional
density are consistent with the length of the DNA extension.
Beyond identifying the upstream DNA sequence at position
?66, this additional DNA density extending from lobe A also
provided a marker for the position of the TATA box, which is
36 bp from the end of this extended DNA, and thus within lobe
A of the rearranged state (Figure 4A).
Wefurther examined theorientation of promoter DNA onTFIID
by covalent labeling of the SCP DNA with 1.4 nm maleimido-
Nanogold at either ?36 (TATA box) or +45 on the SCP promoter
sequence. This strategy takes advantage of the high contrast of
Nanogold relative to that of protein and DNA in transmission EM,
tive to protein and DNA density (see Extended Experimental
Procedures). Analysis of the 2D reference-free class averages
for TFIID-TFIIA-SCP(+45 gold) revealed that the downstream
region of theSCP isbound bylobe C(Figure 4B). Moreover, con-
TFIID-TFIIA-SCP(TATA gold) demonstrated that the upstream
DNA sequence is bound by TFIID within lobe A at a location
across the central channel from lobe C (Figure 4C).
TFIIA Localizes to Lobe A in Both the Canonical and
Rearranged Conformations of TFIID
We next investigated the location of TFIIA in the rearranged
TFIID-TFIIA-SCP complex by analyzing TFIIA that was labeled
with 1.8 nm Ni2+-NTA-Nanogold. After conducting 2D refer-
ence-free analysis of the high-defocus cryo-EM particles, we
applied the determined alignment parameters to the low-defo-
cus focal mate and calculated the corresponding class aver-
ages. Nanogold densities identified the TFIIA binding site within
lobe A of the rearranged state (Figure 4D), adjacent to the TATA
Nanogold location (Figure 4C). Importantly, the analysis of 2D
reference-free class averages corresponding to the canonical
state also demonstrated that TFIIA localized to lobe A in its
alternate configuration relative to the BC core (Figure 4E). These
findings are in agreement with the well-characterized direct
interaction of TBP and TFIIA, and further demonstrate, in the
context of TFIID, that TFIIA interacts with the complex at a loca-
tion close to the TATA box-binding site. These findings therefore
suggest that both TBP and TFIIA are localized within lobe A
during the structural transition between canonical and rear-
ranged states of TFIID.
Presence or Absence of TFIIA
The cryo-EM data of the rearranged conformation of the TFIID-
TFIIA-SCP complex suggest that the downstream DNA core
promoter elements MTE and DPE are bound by lobe C, whereas
the TATA box and possibly the Inr are bound by lobe A. To test
and extend this model, we performed footprinting analyses of
the TFIID-SCP complex in the presence or absence of TFIIA.
DNase I footprinting of TFIID-SCP showed an extended region
footprinting data for TFIID-SCP (Figure5A;Juven-Gershon etal.,
2006). The addition of TFIIA did not alter the downstream in-
teractions of TFIID with the Inr, MTE, and DPE core promoter
elements, but it did result in a substantial increase in the binding
of TFIID to the TATA box and flanking DNA sequences from ?38
to ?20 (Figures 5A, S4A, and S4B). DNase I footprinting analysis
of both DNA strands in the absence or presence of TFIIA
revealed distinct patterns of protection and cleavage throughout
the core promoter region. The results indicate that the DNA
sequence between the Inr and MTE/DPE sites exhibits a phasing
in DNase I sensitivity in which one face of the SCP DNA between
Inr and MTE-DPE is susceptible to DNase I cleavage, whereas
the opposite face remains protected (Figure 6). Furthermore,
the accessible face of the DNA exhibits DNase I hypersensitivity
upon binding of TFIID (Figure 5A).
To obtain high-resolution data on the interactions of TFIID
with the core promoter, we carried out footprinting analyses
with methidiumpropyl-EDTA-Fe(II) (MPE-Fe), an intercalating
agent that delivers Fe(II) for oxidation of the deoxyribose phos-
phate backbone of DNA and provides single-basepair resolution
of protein-DNA contacts (Hertzberg and Dervan, 1984; Papa-
vassiliou, 1995; Van Dyke et al., 1982). The MPE-Fe footprinting
data obtained for TFIID-SCP and TFIID-TFIIA-SCP reveal the
extensive and continuous interaction of TFIID with DNA from
the DPE through the Inr, as well as the TFIIA-dependent pro-
tection of 8 bp (?31 to ?24) of DNA encompassing the TATA
box (Figures 5B, S4C, and S4D). The strong stimulation of TFIID
binding to the TATA box is consistent with the results of
previous studies on the binding of TBP to DNA (Geiger et al.,
1996; Kim et al., 1993a, 1993b; Nikolov et al., 1995). The region
of DNase I protection observed on only one face of the helix
between the MTE/DPE and Inr also shows continuous pro-
tection from MPE-Fe(II) cleavage on both DNA strands. This is
likely due to inhibition of the DNA unwinding (which is neces-
sary for MPE intercalation) that would occur as a result of pro-
tein being bound to one side of the helix (Uchida et al., 1989).
Thus, the DNase I and MPE-Fe footprinting results (summarized
in Figures 6A and 6B; Movie S2) provide insight into TFIID-
DNA contacts that complements the cryo-EM data and contrib-
utes to the placement of the core promoter DNA on the TFIID
To explore the effect of core promoter architecture on
TFIID-promoter interactions, we performed DNase I footprinting
experiments with mutant SCP DNA constructs in the presence
or absence of TFIIA (Figure 5C). Mutation of the TATA box
within the SCP sequence (mTATA) resulted in a wild-type inter-
action with the promoter DNA from the Inr to the DPE, as seen
previously (Juven-Gershon et al., 2006). In addition, the inclu-
sion of TFIIA resulted in a weak but detectable footprint over
the mutant TATA box (Figure S5). The strong resemblance
between the mTATA and the wild-type SCP DNase I protection
patterns suggested that TFIID is bound to the mTATA sequence
in the rearranged conformation. To test this hypothesis, we
collected cryo-EM data and visualized a sample of TFIID-
TFIIA-SCP(mTATA) (Figure S6). This experiment revealed that
TFIID binds to the mTATA promoter in a conformation nearly
identical to that observed with the wild-type SCP sequence.
Thus, the combined footprinting and EM data indicate that the
Cell 152, 120–131, January 17, 2013 ª2013 Elsevier Inc. 125
conformation for the SCP and mTATA promoter architectures.
We further addressed the conformation of promoter-bound
TFIID by analyzing promoters that contain mutations in the Inr
Dictates TFIIA-Dependent and -Indepen-
dent Interactions of TFIID with Core Pro-
(A and B) DNase I (A) and MPE-Fe (B) footprinting
of TFIID-SCP and TFIID-TFIIA-SCP.
(C) DNase I footprinting on wild-type and mutant
SCP DNA sequences. 50-labeled downstream
probes wereanalyzed for DNase Iprotection inthe
TATA (mTATA), Inr (mInr), and MTE/DPE (mMTE/
DPE). See also Figures S4, S5, and S6.
5. Core PromoterArchitecture
(mInr) or MTE/DPE (mMTE/DPE) pro-
moter motifs (Figure 5C). TFIID did not
interact appreciably with either the mInr
promoter or the mMTE/DPE promoter in
the absence of TFIIA, as seen previously
(Juven-Gershon et al., 2006). However,
the addition of TFIIA resulted in strong
binding of TFIID to the TATA box, as
well as to sequences from the Inr through
the DPE regions. Inthe presence of TFIIA,
the overall patterns of protection ob-
served with the mInr and mMTE/DPE
promoters are similar to those seen with
the wild-type SCP. It thus appears likely
that TFIID-TFIIA binds to the mInr and
mMTE/DPE promoters in the rearranged
conformation. Hence, both the footprint-
ing and EM data suggest that TFIID binds
to wild-type and mutant SCPs in the rear-
Promoter-Bound TFIID Is in
a Rearranged Conformation
tional landscape for human TFIID. The
TFIID complex exists in a canonical state
in which lobe A interacts with lobe C,
and a rearranged state in which lobe A is
found in a reorganized position bound to
of TFIID appears to be the form of TFIID
that is bound to core promoter DNA,
whereas the canonical state of TFIID
appears to be the form of free TFIID.
EM-based mapping, together with foot-
printing experiments on promoter DNA,
allowed us to define the approximate
locations of the core promoter elements,
as well as the TFIIA cofactor, on the rearranged form of TFIID.
The architecture of the rearranged conformation of TFIID may
be an important component in the assembly of the preinitiation
126 Cell 152, 120–131, January 17, 2013 ª2013 Elsevier Inc.
Our visualization of the rearranged TFIID-TFIIA-SCP complex
provides structural insight into the isomerization of human TFIID
as proposed previously by Chi and Carey (1996), Emami et al.
(1997), and Lieberman and Berk (1994). These investigators
probed TFIIA- and activator-dependent interactions between
human TFIID and downstream promoter regions in which down-
stream DNA contacts developed only after TFIID was recruited
to the promoter through TBP-TATA interactions. On the basis
of this observation, TFIID was proposed to undergo an isomeri-
zation as it created TAF-DNA contacts away from the original
site of interaction at the TATA box. Although the authors did
not have any information about the structure of TFIID at that
time, we can now explain their biochemical footprinting data
given the interplay between TFIID’s structural dynamics and
promoter binding that we observed in our cryo-EM experiments.
This is an example of how structural analysis can provide funda-
mental mechanistic insight and serve as a framework to clarify
Our findings have resulted in distinct conclusions relative to
those obtained in earlier work on the binding of yeast TFIID to
DNA (Elmlund et al., 2009; Papai et al., 2010). When probed
with DNase I footprinting (Sanders et al., 2002), yeast TFIID
only makes TATA box contacts (<10 bp). Perhaps because of
this small contact region, previous EM studies of yeast TFIID
were unable to localize clearly the promoter DNA bound to the
structure, which appears to correspond to the canonical confor-
mation of TFIID. In contrast, our work with the rearranged human
TFIID bound to the SCP allowed us to visualize unambiguously
large and continuous densities for promoter DNA (50 bp of
DNA across the central channel of TFIID from lobe C to lobe A,
and 20 bp of upstream DNA exiting lobe A). Our direct observa-
tion of DNA bound by human TFIID, in combination with our gold
Figure 6. Structural Description of the Re-
arranged TFIID-TFIIA-SCP Complex Rela-
tive to the TSS
Promoter DNA for SCP(?66) was docked into the
TFIID-TFIIA-SCP(?66) map (shown in mesh).
(A and B) DNA models were colored based on
protection patterns for DNase I and MPE-Fe. Red
and pink surfaces indicate cleavage, the blue
surface indicates partial cleavage, and lack of
surface indicates protection. Asterisk (*) in (B)
indicates a DNase I hypersensitive site at +3.
Black lines in (B) indicate regions of continuous
protection along SCP helix.
(C) The proposed location of the crystal structures
of TBP-TFIIA-TFIIB on TATA box DNA is in close
proximity to Inr (Protein Data Bank ID codes 1VOL
and 1NVP). The unresolved DNA path between Inr
and TATA is indicated by a dotted line. See also
labeling and footprinting studies, pro-
vides a detailed structural model for
TFIID’s 100 bp footprint on promoter
DNA. We speculate that the accessibility
of DNA segments near the transcription
start site (TSS) within the rearranged
conformation of TFIID provides a model of promoter-bound
TFIID that may be primed for RNAPII loading at the TSS.
It is possible that the structural dynamics of lobe A described
here may be present in all eukaryotic forms of TFIID. The struc-
tural differences observed between previous 3D models of yeast
and human TFIID (Papai et al., 2011) may be due, at least in part,
to the structural plasticity of the TFIID complex. Nevertheless, it
is possible that these structural differences may reflect true
divergence among species, because metazoan core promoter
motifs such as the MTE and DPE do not appear to be present
Theextremely flexibleattachment oflobeAwithin TFIIDposed
a significant challenge in our attempt to determine the structure
of human TFIID bound to promoter DNA (Figures 3 and 4). The
underlying structural transitions explain why our earlier attempts
to obtain a 3D reconstruction of TFIID-TFIIA-SCP using a single
canonical model failed to provide a stable 3D structure (data not
shown). Only after rederiving two distinct starting models using
ab initio single-particle EM methods were we able to describe
3D models corresponding to both the canonical and rearranged
conformations, with the latter corresponding to the DNA-bound
form of the complex. Even after these two predominant confor-
mations are taken into account, the resolution of the structures
is limited by the flexibility of lobe A within TFIID, which is present
in both conformations (Figure 2). This flexibility, combined with
the presence of stabilizing agents (detergent, sugar, and glyc-
erol) within the sample buffer and the use of a carbon film as
support (both of which decrease the contrast of the images),
has so far prevented the accurate particle alignments that are
necessary for high-resolution refinement of the data. The advent
of technical developments that will ultimately improve the signal-
to-noise ratios in cryo-EM images (e.g., direct electron detection
Cell 152, 120–131, January 17, 2013 ª2013 Elsevier Inc. 127
and phase-plate implementations) may enable investigators to
obtain the contrast necessary to partition TFIID into specific
conformations to achieve higher resolution.
Interaction of Human TFIID with Promoter DNA
Lobe C Interacts with Downstream Promoter DNA
We have developed a model to describe the DNA path through
the modular subdomains of the TFIID-TFIIA-SCP complex (Fig-
ures 6A, 6B, and 7C; Movie S2). Gold labeling of the SCP DNA
at the +45 position indicated that the density extending off of
lobe C is the downstream promoter DNA (Figure 4B). It thus
appears that the MTE and DPE promoter elements are bound
by lobe C (Figures 6A and 6B), which suggests that lobe C
contains subunits TAF6 and TAF9 (Figures 7C and 7F; Burke
and Kadonaga, 1997; Theisen et al., 2010). The presence of
DNA on the surface of lobe C is consistent with the DNase I
footprinting data, which revealed that one face of the down-
stream promoter DNA is protected, whereas the other face is
exposed to solution and accessible for cleavage by DNase I
(Figures 6A and 6B, middle). On each strand between the Inr
and MTE, there is a distinct 10 bp phasing of DNase I hypersen-
sitive sites at ?2, +18, and +28 (with probe DNA 50-labeled
upstream) and at +4 and +13 (with probe DNA 50-labeled down-
stream). This periodic DNase I digestion pattern is consistent
with the promoter DNA being rotationally positioned on the sur-
face of TFIID.
Lobe A Contacts Promoter DNA Upstream of the TSS
Across the central channel from lobe C, the flexibly attached
lobe A interacts with SCP DNA extending from Inr to the TATA
box (Figures 6A, 6B, and 7C). Previous work demonstrated
that this region of the core promoter interacts with a TAF1,
TAF2, and TBP subcomplex that is also able to direct transcrip-
tion from TATA-Inr promoters (Chalkley and Verrijzer, 1999).
Given the modularity of lobe A within the context of TFIID, it is
likely that lobe A contains TAF1, TAF2, and TBP, and serves as
a modular domain of TFIID that is capable of interacting with
promoter DNA upstream of the TSS (Figure 7).
This proposed composition of lobe A is consistent with a
study by Wright et al. (2006) that addressed the integrity of the
TFIID complex in vivo. Through systematic RNA-interference-
mediated knockdown, the authors defined a stable TAF4, ?5,
?6, ?9, and ?12 subcomplex nucleated by TAF4. In contrast,
knockdown of TAF1, TAF2, TAF11, or TBP did not affect the
integrity of the TFIID complex, suggesting a peripheral location
within TFIID. Thus, lobe A likely comprises TAF1, TAF2, TAF11,
and TBP, existing as a modular domain of TFIID, whereas a
stable TAF4, ?5, ?6, ?9, and ?12 subcomplex may correspond
to the BC core (Figure 7).
To model the DNA path through lobe A, we incorporated two
bends within lobe A to accommodate the 120?angle that TFIID
imposes on the downstream and upstream DNA regions (Fig-
ure 6B). Initial attempts to trace the DNA path through the
TFIID-TFIIA-SCP structure using a single bend at the TATA box
failed to generate a model that was compatible with the experi-
mentally obtained positions of downstream and upstream DNA
locations (data not shown). Given the angle of upstream DNA
and the location of the ?66 position, we modeled the location
of TBP and TATA box DNA to be 36 bp (12.2 nm) downstream
from the ?66 position observed in the TFIID-TFIIA-SCP(?66)
structure. We propose that, after this bend is incorporated at
?31/30, a second, gradual bend forms along the DNA between
the TATA box and Inr, and that this DNA deformation between
the TATA box and Inr could play a role in positioning TBP and
TFIID Interacts with Diverse Promoter Architectures
through the Rearranged Conformation
Because the majority of promoters within the Drosophila and
human genomes do not contain all four of the core promoter
motifs engineered into the SCP (Juven-Gershon and Kadonaga,
2010), we analyzed promoter architectures with fewer core
promoter motifs to investigate the relevance of the rearranged
TFIID-TFIIA-SCP structure. To that end, we compared TFIID in-
teractions withwild-type versus mutantversions of theSCP (Fig-
ure 5C). These data suggest that TFIID interacts with TATA-Inr,
Figure 7. Model of TFIID’s Interaction with
Core Promoter DNA in a Conformation-
and TFIIA-Dependent Fashion
(A–C) Conformations adopted by TFIID-TFIIA.
(D–F) Conformations adopted by TFIID alone.
(A and B) TFIIA stabilizes TFIID in the canonical
(C) The addition of SCP DNA stabilizes the re-
arranged conformation for the ternary complex
canonical and rearranged states equally.
(F) Upon addition, SCP DNA is bound by TFIID in
the rearranged conformation. Brackets ([ ]) denote
that (F) was observed only through biochemical
128 Cell 152, 120–131, January 17, 2013 ª2013 Elsevier Inc.
TATA-MTE/DPE, and Inr-MTE/DPE promoters in the rearranged
conformation (Figure 7).
With both the wild-type SCP and the three mutant versions
of the SCP (mTATA, mInr, and mMTE/DPE), we observed that
TFIIA stimulates the binding of TFIID to the TATA box region
(Figure 5C). This effect is consistent with the well-established
TFIIA-mediated enhancement of TBP binding to the TATA box
(Thomas and Chiang, 2006). With the mTATA promoter, the
primary interaction of TFIID with the DNA is via the Inr, MTE,
and DPE motifs, and a weak stimulation by TFIIA of the binding
of TFIID to the mutant TATA box region is also observed. With
the mInr and mMTE/DPE promoters, it seems likely that TFIIA
stimulates the binding of TBP to the TATA box, and that the
remainder of the TFIID complex then interacts with the Inr
through the DPE region of the core promoter, irrespective of
the presence of consensus Inr or MTE/DPE elements. These
findings may be analogous to the previously observed stimula-
tion of the binding of partially purified TFIID to the downstream
promoter region of the adenovirus major late promoter (which
lacks MTE/DPE motifs) by the upstream stimulatory factor
(USF; Sawadogo and Roeder, 1985; Van Dyke et al., 1988). In
this light, it is possible that other sequence-specific activators,
as well as coactivators, may function in a related manner to
stabilize TFIID on promoter DNA and thus promote the formation
of the rearranged conformation.
We believe that our description of the conformational land-
scape of TFIID provides a conceptual framework for under-
standing the functional interactions that occur between TFIIA
and core promoter architecture on the TFIID structure. In partic-
ular, the dynamic conformational landscape of TFIID may have
regulatory consequences within the cell by providing specific
structural targets that can be recognized by transcriptional acti-
vators and repressors.
Full details of the experimental procedures are presented in Extended Exper-
Protein and Nucleic Acid Preparation
Purification of human TFIID and TFIID-TFIIA-SCP was performed as described
extract was subjected to immunoprecipitation with a a-TAF4 monoclonal anti-
body (mAb; BD Biosciences). Assembly of the TFIID-TFIIA-SCP complex
involved addition of a 103 excess of recombinant TFIIA (Sun et al., 1994)
and SCP DNA (IDT) prior to elution from the resin (Figure S7).
The DNA sequence used for the SCP was taken directly from the originally
published sequence of SCP1 (Juven-Gershon et al., 2006): GTACTTATATAA
AGCAGACGTGCCTACGGACCG. For SCP(?66), the following sequence was
added immediately upstream of the SCP sequence: CTCGCGCCACCTCTG
Mutant promoter sequences were taken directly from a previous mutational
analysis of SCP1 (Juven-Gershon et al., 2006).
Sample Preparation and EM
For negative-stain microscopy, 4 ml of TFIID sample was directly applied to
glow-discharged holey carbon film covered with a continuous thin-carbon
support on a 400 mesh copper grid (Electron Microscopy Sciences). For
cryo-EM, samples were vitrified using a Vitrobot (Gatan) that was set to
100% humidity at 4?C. Then 4 ml of sample was incubated for 30 s to 1 min
on a carbon-thickened C-flat grid (Protochips) with 4 mm holes (spaced 2 mm
apart, with a thin-carbon support within the holes), blotted for 6.5 s, and
then plunge-frozen in liquid ethane.
The negative-stain data shown in Figure 2 were collected using a Tecnai
T12 bio-TWIN transmission electron microscope operating at 120 keV under
low-dose conditions (15 e?/A˚2) on Kodak SO163 film at a nominal magnifica-
tion of 30,0003 from defocuses ranging from ?0.70 mm to ?1.20 mm. Micro-
graphs were digitized in a Nikon Super Coolscan 8000 at a pixel size of
12.7 mm (4.23 A˚/pixel). Cryo-EM data were collected on a Tecnai F20 TWIN
transmission electron microscope operating at 120 keV using a dose of
25 e?/A˚2on a Gatan 4k 3 4k CCD at a nominal magnification of 80,0003
(1.501 A˚/pixel). Leginon software was used to automatically focus and collect
exposure images (Suloway et al., 2005).
Image Preparation and Analysis
Particles from the negative-stain data shown in Figure 2 were manually picked
using Boxer (EMAN; Ludtke et al., 1999), contrast transfer function (CTF) esti-
mated using CTFFIND3 (Mindell and Grigorieff, 2003), and phase flipped using
SPIDER (Frank et al., 1996). Cryo-EM data were prepared using the Appion
image processingenvironment (Lander etal.,2009)where particles wereauto-
matically picked using Signature (Chen and Grigorieff, 2007), CTF estimated
using CTFFIND3 (Mindell and Grigorieff, 2003), phase flipped using SPIDER
(Frank et al., 1996), and normalized using XMIPP (Sorzano et al., 2004).
For negative staining, we performed a 2D reference-free image analysis
using an iterative routine implementing a topology-representing network
(Ogura et al., 2003) in combination with multireference alignment within
IMAGIC (van Heel et al., 1996). For cryo-EM, 2D reference-free image analysis
was performed exclusively within IMAGIC (van Heel et al., 1996) through iter-
ative rounds of multivariate statistical analysis and multi-reference alignment.
We performed 3D refinements on phase-flipped particles using an iterative
projection matching and 3D reconstruction approach employing libraries from
EMAN2 and SPARX software packages (Hohn et al., 2007; Tang et al., 2007).
We then refined 34,167 particles from cryo-EM grids prepared from the TFIID-
TFIIA-SCP sample against low-pass-filtered models of the canonical and rear-
ranged state. For TFIID, we collected and refined 30,800 particles. After
completing this global projection matching routine, we further refined all
data in FREALIGN (Grigorieff, 2007) and low-pass filtered them at the final
resolution while applying a negative B-factor using bfactor.exe.
Maps have been deposited in the Electron Microscopy Data Bank (EMDB)
under accession numbers 10832 (TFIID-TFIIA-SCP rearranged), 10834 (TFIID-
TFIIA-SCP canonical), 10835 (TFIID rearranged), and 10839 (TFIID canonical).
figures, and two movies and can be found with this article online at http://dx.
We thank members of the Nogales laboratory for constant advice and help
with EM analysis; R.J. Hall for initial work on this project; A. Leschziner for
advice on the implementation of OTR; S. Zheng for providing TAF4 mAb; D.
King for providing peptides; and R. Tjian, W.L. Liu, and R. Coleman for critical
National Institutes of Health (GM041249 to J.T.K. and GM63072 to E.N.) and
the Human Frontier in Science Program (RGP0039/2008 to E.N.). E.N. is a
Howard Hughes Medical Institute Investigator.
Received: June 20, 2012
Revised: September 20, 2012
Accepted: November 28, 2012
Published: January 17, 2013
Cell 152, 120–131, January 17, 2013 ª2013 Elsevier Inc. 129
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