(Received for publication, July 16, 1996, and in revised form, October 25, 1996)
From the Department of Biological Chemistry, University of California, Los Angeles, California 90095-1737
Transcription factor (TF) IIA performs two
important regulatory functions during RNA polymerase II transcription:
it is required for efficient binding of TFIID to a core promoter and it
mediates the effects of upstream activators, both through direct
interaction with the TATA box binding protein (TBP). To begin studying
how TFIIA mediates these effects, we used a highly sensitive protease footprinting methodology to identify surfaces of human TFIIA
participating in TFIIA·TBP·TATA ternary complex formation.
Chymotrypsin and proteinase K cleavage patterns of TFIIA bearing a
32P-end-labeled subunit revealed that amino acids
59-73 were protected from cleavage both in the context of an
immobilized ternary complex and in a binary complex with TBP alone. In
contrast, amino acids 341-367 in the
portion of a
32P-labeled
-
subunit were protected in the ternary
but not in the binary complex, implying that those residues interact
with promoter DNA. The regions of human TFIIA identified by protease footprinting are homologous to and encompass the yeast TFIIA residues that contact TBP and DNA in the recently solved crystal structure of
the yeast ternary complex. The conservation of the regions and residues
mediating complex formation implies that yeast and human TFIIA employ
the same mechanism to stabilize the binding of TFIID to a core
promoter.
Transcription of protein-encoding genes by RNA polymerase II (pol
II)1 is regulated by an intricate array of
protein-protein and protein-DNA interactions (1, 2, 3, 4). Understanding how
these interactions mediate formation of a transcription complex over a
core promoter is a central problem in the field of gene expression. In
the step-wise model, transcription complex assembly is nucleated by
binding of the general initiation factors (ranscription
actor) TFIID and TFIIA to the TATA box generating the
"DA complex" (5). The parallel between the ability of gene
activators to facilitate DA complex formation and to activate
transcription suggests that the complex plays a key role in regulation
(6).
TFIID is a multisubunit complex consisting of TATA box binding protein (TBP) and eight or more TBP-associated factors (TAFs) (7, 8, 9, 10). TBP alone, when it is substituted for TFIID, can nucleate the formation of a basal transcription complex that is fully functional but not responsive to activators. In contrast, TAFs are thought to act as co-activators because they are dispensable for basal transcription but are required to obtain activator-responsive transcription in vitro. In addition to TAFs, a fraction called upstream-factor stimulatory activity (USA), which contains both positive and negative co-regulators, potentiates transcriptional activation (11).
In the DA complex, TFIIA functions both to stabilize the relatively weak binding between TFIID and the TATA box and is necessary for activator recruitment of TFIID (12, 13, 14); its role in recruitment depends on the TAFs since activators have no effect on TBP·TFIIA complex formation. In the stepwise model, the formation of the transcription complex is completed by the successive association of TFIIB, RNA polymerase/TFIIF, TFIIE, and TFIIH (2, 4). An alternative model for complex formation involves a holoenzyme (15) containing RNA polymerase II and many of the other general initiation factors. The holoenzyme was discovered initially in yeast and more recently in mammalian cells. Both the yeast and some mammalian versions have been reported to lack TFIID and TFIIA (16, 17, 18). One possible function of the DA subcomplex is to form an activator-responsive platform for recruitment of the holoenzyme.
Human (and Drosophila) TFIIA is a multisubunit protein
consisting of three subunits called (LN),
(LC), and
(S)
(19, 20, 21, 22, 23, 24, 25).
and
are synthesized as a precursor that is processed
proteolytically to generate the mature subunits although the
unprocessed form is functional in vitro. Yeast TFIIA
consists of only two subunits, TOA1 and TOA2 (26, 27). TOA1 is
homologous at its amino and carboxyl termini to regions of the human
and
subunits, respectively, and TOA2 is homologous to the human
subunit (19, 20, 21, 22). Analysis of systematic internal deletion mutants
of both subunits demonstrated that all of the regions conserved between
yeast and human TFIIA were required for yeast viability (28). For TOA1 (
and
), these deletion mutants defined the amino- and
carboxyl-terminal ends as essential but the nonconserved, middle region
as dispensable for viability. A deletion mutant removing residues
217-240 abolishes binding to TBP. For TOA2 (
), all of the internal
deletions but one resulted in inviable yeast, making it difficult to
genetically define functional domains. Alanine scanning mutants of both
subunits were screened for temperature-sensitive (ts) growth
phenotypes. In TOA1, all the ts mutations were in basic residues
between residues 253 and 259. These mutants bound normally to TBP but
could not form complexes, implying a decreased ability to bind DNA.
Among the three ts mutants identified in TOA2, one double mutant,
D73A/D74A, bound less tightly to TBP although complex formation was
unaffected, and the other two exhibited no detectable difference in
their ability to bind TBP or form ternary complexes. Human TFIIA
has also been analyzed by alanine substitution mutations and Y65A (equals
Y69 in yeast TOA2) caused the greatest decrease in the ability of IIA
to stabilize TBP binding (29).
The crystal structure of the ternary complex containing the yeast
(y)CYC1 TATA box, yTBP, and yTFIIA was recently published (30, 31). In
the crystal structure, yTBP, which encodes two 80-amino acid direct
repeats, folds into a pseudosymmetric saddle-like shape containing a
hydrophobic concave surface composed of a 10-strand antiparallel
-sheet, which interacts with the minor groove of the DNA. TBP
binding distorts the TATA box by inserting phenylalanine residues
between the first and final base pairs of the recognition site,
resulting in bending of the DNA by 80° and broadening of the minor
groove (32, 33). yTFIIA, on the other hand, is composed of a four-helix
bundle and a 12-strand
-barrel (30, 31). The
-barrel is made of
six strands each from the carboxyl terminus of TOA1 (TOA1C~human
TFIIA
) and the carboxyl terminus of TOA2 (
). The four-helix
bundle is composed of the amino terminus of TOA1 (TOA1N~human
TFIIA
) and the amino terminus of TOA2. The interaction between TFIIA
and TBP places the
-sheets of TOA2 and TBP in close proximity,
resulting in a continuous 16-strand
-sheet. TFIIA interacts with DNA
through basic residues in TOA1C that lie within a loop connecting two
-strands.
DNA-protein cross-linking studies have confirmed certain aspects of the
structure in solution (34, 35). Collectively, these studies show
cross-linking was observed between DNA and upstream of the TATA
box, between
and both sides of the TATA box, and with
at one
position upstream of the TATA. In the crystal structure of the yeast
complex, only a region homologous to part of the
subunit contacts
DNA.
To study how TFIIA stabilizes TFIID/TBP binding, we investigated the
assembly of the ternary complex containing a mammalian TATA box, human
TBP, and human TFIIA. Presently, there is no genetic analysis of the
-
subunit of human TFIIA or crystal structure of free TFIIA. To
identify the interactions mediating complex formation, the differences
between free TFIIA and complexed TFIIA were characterized using
protease footprinting. The interactions of TFIIA with TBP and DNA were
identified along with the accessible regions of TFIIA. The results of
our protease footprinting studies are placed within the context of the
recently solved x-ray structure of the yeast ternary complex.
To engineer vectors containing both His and
HMK tags at alternate termini, pET21a-N-HMK was constructed by cloning
an oligonucleotide, 5-TATGCGCCGCGCCAGTGTGGGATCCC-3
, encoding the HMK
phosphorylation site (HMK tag) between the NdeI and
XhoI sites of pET21a (Novagen). pET11d-N-His/C-HMK was
constructed by cloning an oligonucleotide, 5
-CTAGCCACCACCACCACCACCACG-3
, encoding the His tag between the NheI and BamHI sites of pET11d-C-HMK (36). A
BamHI fragment encoding TFIIA
-
and a
BamHI-BglII fragment encoding TFIIA
coding
regions were synthesized by PCR and ligated into the BamHI site of pET11d-N-His/C-HMK and pET21a-N-HMK, respectively, using T4
ligase. pN-HMK-TFIIA
81 was made by digesting
pN-HMK-TFIIA
-C-His at the EcoRI site within the coding
region, repairing with the Klenow fragment and deoxynucleoside
triphosphates, and inserting a stop codon oligonucleotide,
5
-CTAATCTAGATTAG-3
, using T4 DNA ligase. pC-HMK-TFIIA
-
38,
129,
254 were constructed by deleting the coding region between
the NheI and ScaI (
38), NdeI
(
129), and MscI (
254) sites within the coding regions,
respectively, by digesting with these restriction enzymes, filling in
with the Klenow fragment, and ligating using T4 DNA ligase. These
constructs encode internal deletions between residues 3 and 38, 129 and
254, respectively.
TFIIA and GST-TBP (The GST-TBP
expression vector was a generous gift of Arnie Berk, UCLA) were
purified as described (20, 37). TFIIA containing the HMK tag on the
carboxyl terminus of the -
subunit was further purified by
affinity chromatography over a GST-TBP affinity resin (28). The TFIIA
deletion constructs were transformed into BL21(DE3) and inoculated into
250 ml of Luria broth containing 50 µg/ml ampicillin. When the cell
culture reached an A600 of 0.6, protein
expression was induced for 1.5 h by the addition of
isopropyl-1-thio-
-D-galactopyranoside to 0.3 mM. All of the following steps were performed at 4 °C.
The cells were collected by centrifugation in a Sorvall SA600 rotor at
5,000 rpm for 5 min, washed with Buffer A (20 mM HEPES, pH 7.9, 0.2 mM EDTA, 0.5 M KCl), collected again
by centrifugation at 5,000 rpm for 5 min, resuspended in 50 ml of
Buffer A containing 0.5 mM DTT, 0.5 mM PMSF, 50 µg/ml benzamidine, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A,
and lysed by sonication. For TFIIA
81 and for TFIIA
-
38
and
129, the membrane fraction was extracted with 30 ml of Buffer D
(20 mM HEPES, pH 7.9, 0.2 mM EDTA, 20% glycerol) containing 0.2 M KCl, 8 M urea, 0.5 mM DTT, and 0.5 mM PMSF. The urea was removed
by step-wise dialysis into Buffer D containing 0.3 M KCl,
0.5 mM DTT, and 0.5 mM PMSF and by decreasing concentration of urea from 2 to 0.5 to 0 M. For
TFIIA
-
254, the lysate was precipitated by successive
additions of 0.20, 0.25, and 0.30 g/ml of
(NH4)2SO4, stirring for 30 min, and
then collecting the precipitate by centrifugation at 10,000 rpm for 10 min. TFIIA
-
254 was in the 0.25 g/ml precipitate and was
solubilized in 3 ml of Buffer D containing 0.3 M KCl, 0.5 mM DTT, and 0.5 mM PMSF. Recombinant human TBP
was a generous gift from Zong Juo and Richard Dickerson (UCLA)
(38).
TBP-TFIIA-TATA box
ternary complexes were formed in a 50-µl mixture containing 3 pmol of
biotinylated adenovirus E4 DNA template, a synthetic biotinylated
oligonucleotide (biotin-5-GGATCCCCAGTCCTATATATACTCGCTCTGC-3
) immobilized on streptavidin-conjugated M-280 magnetic beads (Dynal), with 500 ng of TBP, and 400 ng of 32P-labeled TFIIA. After
30 min at 30 °C, the complexes were washed 3 times with 100 µl of
binding buffer (12 mM HEPES, pH 7.9, 12% glycerol, 60 mM KCl, 2 mM MgCl2, 0.02% Nonidet
P-40, 0.5 mM DTT) and digested with 5-200 ng of either
proteinase K (Promega) or chymotrypsin (Sigma)
alongside free TFIIA or TFIIA incubated with immobilized DNA. TFIIA
incubated with immobilized DNA was only washed once to maintain some of
the protein in the presence of the resin for the control reaction.
After 1.5 min, the cleavage reactions were terminated by addition of
PMSF to 10 mM and freezing in dry ice. SDS-loading dye was
added, and the cleavage products were fractionated on 15% total
acrylamide-3.3% cross-linker or 16.3% total acrylamide/5.0%
cross-linker tricine-SDS gels. Binary complexes were formed and
footprinted as described (36). Protease sequencing ladders were
generated by denaturing 120 ng of labeled TFIIA in 10 mM
HEPES, pH 7.9, 10% glycerol, 50 mM KCl, 2 M
urea, 1 mM DTT, and 0.1% SDS at 50 °C and digesting at
37 °C with 3 µg of endoproteinase Lys-C (1-10 min), 1.5 µg of
clostripain (5-90 min), or 5 µg endoproteinase Glu-C (1-10 min)
(Promega). The cleavage sites were assigned using a combination of the
known amino acid sequence, prestained molecular weight markers
(Bio-Rad), and deletion markers. The positions of the digestion
products in the experimental lanes were mapped using all of these
markers and a semi-log plot of mobility based on these markers. The
precision of the semi-log plot in the protected regions is ±2
residues. The gels were autoradiographed by exposure to XAR-5 film. The
autoradiographs were scanned into Adobe Photoshop 3.0 using a
ScanMakerIII (Microtek), imported into Microsoft PowerPoint 4.0, and
labeled and printed onto glossy paper on a Tektronix printer. For
quantitation, either the autoradiographs were scanned with a laser
densitometer or the gel was exposed to a PhosphorImager screen and
quantitated using ImageQuant software (Molecular Dynamics). Sample
lanes in which the extent of digestion was similar, as judged by the
percentage of full-length material remaining in the experimental and
control lanes, were compared, and the ratio of these signals was used
to normalize other signals. To determine the extent to which each band
was protected, the signal ratio of a digestion product in the
experimental lane to that of the control lane was calculated and then
normalized using the signal ratio of full-length protein.
Protease footprinting represents a potentially powerful technology for examining protein-protein and protein-DNA interactions. In its simplest form, the concept is analogous to DNase I footprinting except that, rather than employing nucleases to map a protein binding site on a 32P-end-labeled DNA fragment, proteases are used to map protein contacts on a 32P-end-labeled target protein. The advantage of protease footprinting over deletion and point-mutation analyses is that interactions are studied in the context of a largely native wild-type protein, bypassing the potential for deleterious effects that mutants might have on the overall structure. Furthermore, because the complexed protein is compared with the free protein, conformational changes upon interaction may also be detected quite readily.
In a previous study, we employed protease footprinting to map the binding sites of the viral activator VP16 and Drosophila TAFII40 on human TFIIB (36). The VP16 binding sites correlated with those defined by mutational studies (39), whereas the TAF40 binding site was not previously identified. The subsequent crystal structure of the TBP-TFIIB-TATA box ternary complex (40) revealed that the VP16 and the TAF40 binding sites defined by protease footprinting formed a solvent-accessible wedge and were in close proximity, consistent with their ability to mediate complex formation (41).
Our current study characterizes the interactions of human TFIIA within the TFIIA-TBP-TATA ternary complex to understand how TFIIA assists TBP and TFIID binding to DNA, a step that serves as a key target for transcriptional activators. Although our study was performed independently, comparison with the recently solved crystal structure of the yeast ternary complex validates the utility of the protease footprinting technology for studying intricate protein-protein interactions.
Experimental DesignFig. 1 describes the
protease footprinting procedure used in our study. The TFIIA used for
these experiments contains a unique 32P-end label on one of
its two subunits (-
or
) (Fig. 1A). The 32P-end-labeled TFIIA, the free molecule, or that in the
form of either a binary complex with GST-TBP or a ternary complex with TBP bound to an immobilized adenovirus E4 TATA box oligonucleotide is
digested with a nonspecific protease to generate a nested set of
digestion products (Fig. 1B). The products are fractionated on a high resolution SDS-polyacrylamide gel and autoradiographed to
reveal a ladder (Fig. 1B). The limited proteolysis pattern of TFIIA in the complex is compared with that of free TFIIA to identify
changes in cleavage that occur as a consequence of complex formation.
The loss (or increase) of specific cleavage products is taken as an
indicator of an interaction. To determine whether these footprints are
due to a direct interaction with TBP or the TATA box, protease
footprinting of GST-TBP·TFIIA binary complexes formed in the absence of DNA is compared with the results obtained with
the ternary complexes.
32P-end-labeled TFIIA was generated by constructing
molecules of TFIIA in which one subunit had an HMK tag
(Arg-Arg-Ala-Ser-Val; RRASV) engineered onto one of its termini and a
His tag onto the other for purification using nickel affinity resins
(see Fig. 1A). These molecules were phosphorylated on the
HMK tag using [-32P]ATP and heart muscle kinase; the
labeling was specific as equimolar amounts of a TFIIA molecule
containing untagged subunits incorporated less than 5% of the amount
of 32P generated using tagged subunits (data not
shown).
Fig. 1C shows an example of how the binding reactions were
optimized. Increasing amounts of TFIIA, 32P-end-labeled on
the subunit, were first incubated with a resin containing an
immobilized TATA box and subsequently washed to remove unbound protein.
The bound fraction was electrophoresed on an SDS-polyacrylamide gel and
autoradiographed. Fig. 1C illustrates that specific ternary
complexes were obtained based on the TBP dependence of TFIIA binding
(compare even-numbered lanes with odd-numbered
lanes). At the highest concentration point in this experiment,
34% of the input TFIIA labeled on the
subunit was assembled into
ternary complexes. The HMK-tagged TFIIA molecules used here have been
shown to also be competent to form TATA box-TBP-TFIIA complexes in
electrophoretic mobility shift assays (data not shown).
Fig.
2, A and B, reveals a strong
interaction between a small region of the TFIIA subunit and TBP,
which apparently tethers TFIIA to the ternary complex. Fig.
2A shows chymotrypsin and proteinase K footprints of a
ternary complex containing TFIIA 32P-labeled on the amino
terminus of the
subunit. The chymotrypsin digestion patterns of
free TFIIA (lanes 2-5) or TFIIA in the presence of only the
E4 TATA box oligonucleotide (lanes 11-15) were identical except for a cleavage site that is enhanced in the presence of promoter
DNA (asterisk). In contrast, the cleavage pattern of TFIIA
assembled into a ternary complex (lanes 6-10) revealed a footprint between amino acid residues 51 and 73 as measured by the
Lys-C and clostripain protease sequencing ladders (lanes
28-29, respectively). The position of the Lys-C and clostripain
cleavages were assigned by mapping the known amino acid sequence and
prestained markers against the migration of the digestion products on a
semi-log plot. Proteinase K footprinting revealed that a subset of the same residues (amino acids 59-73) was protected in the ternary complex
(compare lanes 20-23 with lanes 16-19 and
24-27) with concomitant DNA-dependent enhanced
cleavages (asterisks) flanking the protected region. Using
laser densitometry, the three cleavage products between residues 59 and
73 in the proteinase K footprint were 79%, 93%, and 79% protected in
the complex versus the free TFIIA (see "Experimental
Procedures" for normalization procedure).
Protease footprinting of the binary TBP-TFIIA complex (Fig.
2B) demonstrates that the protection described above
represents a direct interaction with TBP. When compared with free TFIIA
(lanes 10-13) or TFIIA incubated with GST alone
(lanes 2-5), proteinase K digestion of a binary complex
between TFIIA and GST-TBP immobilized on glutathione agarose
(lanes 6-9) revealed a footprint. Alignment against the
sequencing ladder and deletion marker (lanes 14-16) revealed that the footprint encompassed the same region (residues 59-73) of the TFIIA subunit as that identified in the ternary complex (Fig. 2A) with the minor difference that the
DNA-dependent enhanced bands were absent. When samples
digested to a comparable extent were quantitated using a PhosphorImager
and compared, there was >90% inhibition of proteolysis of the lowest
three cleavage products on the gel. Taken together, our results suggest
that residues 59-73 of the
subunit mediate a direct
protein-protein interaction with TBP. The residues in human TFIIA
identified by protease footprinting, 59-73, are homologous to residues
63-77 in yeast TOA2, which completely encompasses the TBP-interacting amino acid residues in the crystal structure. We will return to this
point in the discussion (30, 31). The digestion pattern also reveals
that the
subunit is divided into a protease-resistant amino-terminal half, which precludes a protease footprinting analysis of this region, and a more protease-sensitive carboxyl-terminal half
(see Figs. 2A and 2B).
Fig. 3A demonstrates that
TFIIA makes a direct contact with DNA via the portion of the
-
subunit. TFIIA containing
-
32P-labeled at
the carboxyl terminus was assembled into a ternary complex and
footprinted using chymotrypsin. A striking feature of this digest is
the very small number of accessible chymotrypsin cleavage sites, only 5 throughout the length of this subunit. Similar results were obtained
using several proteases. The digestion patterns between free TFIIA
(lanes 11-13) and TFIIA in the presence of the promoter
alone (lanes 3-5), with the exception of background bands
(see pound signs), are identical. However, when assembled into the ternary complex, the three sites nearest the carboxyl terminus
become protected (lanes 7-9). The region identified, residues 341-367 (see Fig. 3A, brackets),
extends from a basic region almost to the carboxyl terminus of the
protein based on the clostripain, Lys-C and Glu-C cleavage sites (see
Fig. 3A, lanes 14-16). Footprints in the same
region were observed using alkaline protease and proteinase K (data
not shown).
The protection of the IIA-
observed in the ternary complex,
unlike the case of the
subunit, is not due to a direct interaction with TBP. This was illustrated by the lack of a footprint in the binary
TBP-TFIIA complex (Fig. 3B); compare, for example, the digestion pattern when the binary complex is formed (lanes
6-9) with that of free TFIIA (lanes 10-13) and TFIIA
in the presence of GST (lanes 2-5). Because no direct
interaction between TBP and IIA
-
can be detected, we conclude
that the protection observed in the ternary complex is a consequence of
an interaction between promoter DNA and TFIIA
-
. This region
contains residues Arg-344, Lys-346, Lys-348, and Lys-350, which are
similar to residues Arg-253, Lys-255, Arg-257, and Lys-259 of yeast
TOA1, that have been shown to contact DNA both in the crystal structure
of the ternary complex (30, 31) and via a genetic analysis (28).
We have identified, using protease footprinting, interactions made
by human TFIIA upon assembly of the TFIIA-TBP-TATA box ternary complex.
Each subunit makes a series of contacts with one of the other
components of the complex, the TFIIA subunit with TBP and the
IIA
-
subunit with DNA. These two interactions allow TFIIA to form
a bracket that stabilizes TBP binding. These results are summarized
schematically in Fig. 4. Together, TBP and TFIIA
function to provide specificity and stability. The preference of TBP
for the TATA box provides the specificity required to bring TFIID to
the promoter, but the weak affinity of TFIIA for DNA is used
advantageously to stabilize binding of TFIID to promoters, which
contain diverse sequences flanking the TATA box.
Correlations with Crystal Structure and Biochemical Data
The
contacts made by TFIIA within the human ternary complex as measured by
protease footprinting are similar to those made within the yeast
ternary complex as identified in the crystal structure. Fig.
5A is a ribbon diagram illustrating the
recently solved crystal structure of the yeast ternary complex (30, 31) in which yTOA1 ( and
), yTOA2 (
), TBP, and the TATA box
oligonucleotide are yellow, green,
blue, and white, respectively. In the structure, the two subunits of yTFIIA are highly intertwined and form a
boot-shaped structure in which TOA2 (
) interacts with TBP and TOA1
(
and
) interacts with DNA. TFIIA binds to the underside of TBP
along the loop (stirrup) (42) connecting
-strand 2 and
-strand 3. A mutagenesis study, in which radical changes were introduced into
surface residues of human TBP, identified an interaction between TFIIA
and residues encompassing the stirrup within the first direct repeat of
TBP, consistent with the crystal structure (43). The ability of these
TBP mutants to support transcriptional activation in vivo
and TBP-TFIIA complex formation in vitro was greatly
diminished.
In Figs. 5B, C, E, and F,
our footprinting results are superimposed on space-filling models of
yeast TFIIA (Fig. 5, B and E) alone and in the
ternary complex (Fig. 5, C and F). Fig. 5, B and C, shows the -TBP interaction using
the same view as in Fig. 5A. Fig. 5B illustrates
yTFIIA alone in which TOA1 (i.e. hTFIIA
-
) is
yellow and TOA2 (i.e. hTFIIA
) is
green. The residues identified by protease footprinting as
interacting with TBP (hTFIIA
residues 59-73 homologous to yTOA2
residues 63-77) are colored red. Of the 15 residues in the
protected region, 11 of the positions are homologous between human and
yeast including all those that directly contact TBP.
In Fig. 5C, TBP (blue) and the TATA box
oligonucleotide (white) have been added to TFIIA. The
protected region in red forms -strands that are part of
the domain that is intertwined with the
-strands of yTOA1 to form
the
-barrel. A comparison of Fig. 5B with 5C
illustrates that the edge of the concave
-sheet region of TBP covers
the "red" region and explains why TBP protects that region from proteolysis.
Fig. 5, D-F, is a separate view of the ternary
complex used to optimize presentation of the region of hTFIIA-
protected in the ternary complex. Fig. 5, E and
F, superimposes the interaction between TFIIA
-
and DNA
identified by protease footprinting onto TFIIA alone (Fig.
5E) and in the ternary complex (Fig. 5F). In this
case, the residues protected in hTFIIA
-
(TOA2) are labeled as
red, and this region lies within the
-strands of the
-portion of hTFIIA
-
. Within the identified region, 16 of the
27 residues are homologous between yeast and human, including 6 basic
ones.
The region of the human TFIIA-
subunit protected from proteolysis
in the ternary complex includes the homologous residues of yeast TOA1
shown to contact DNA directly. In Fig. 5F, when yTBP
(blue) and the TATA box oligonucleotide (white)
are added to yTFIIA (shown from the same view and with the same color
scheme in Fig. 5E), the DNA runs across the
"red" residues, which is consistent with the observed
protease footprint. It appears from the figure that the protected
residue at the bottom of the figure may still be accessible from
underneath. However, the TATA box oligonucleotide used for our
footprinting experiments is longer than the one used in the
crystallography studies, which would increase the protection of that
site.
The one interaction observed in the crystal structure that was not
detected in our study was the insertion of the penultimate tryptophan
residue of yTOA1 into a crevice created where TBP and yTOA2 interact.
Because that tryptophan is the carboxyl-terminal residue in
hTFIIA-
, it is probably too close to our HMK tag for
detection.
Although the protease footprinting analysis does not identify other
interactions of hTFIIA within the ternary complex, it is possible that
a protease-resistant region forms other contacts. The lack of a
proteolytic reagent that cleaves at every surface position does tend to
limit the protease footprinting approach. One possible limitation is
revealed by mutational studies that indicate that an interaction exists
between a highly acidic region of hTFIIA -
and the H2 helix of
TBP (44, 45, 46). We have not identified such a region in our study.
Interactions of TFIIA with TBP in the absence of DNA have been studied
using "GST-pulldown" experiments. Interactions of the ,
, and
subunits (or their homologues) individually with TBP have been
identified. However, in view of the crystal structure, the previous
studies will have to be reevaluated because the subunits are
intertwined and probably will not fold properly on their own. Our
studies define an interaction between only the
subunit and TBP.
A protein-DNA cross-linking study concluded the subunit interacts
with promoter DNA upstream of TBP centered at
45, the
subunit is
close to only
39, and the
subunit can be cross-linked to both
sides of TBP (35). These data are similar to an earlier study (34). We
detect only an interaction between the
portion of the
-
subunit and DNA. The absence of an
-DNA interaction may simply
reflect the use of a TATA box oligonucleotide that is too short to
detect this interaction. It may also be possible that some proportion
of TFIIA undergoes a conformational change within the ternary complex
leading to these other DNA contacts, a finding supported by at least
one recent study (21).
Based on protease sensitivity, it is plausible that other proteins will
interact with TFIIA via the carboxyl terminus of the subunit and
with the
subunit. Because the
subunit binds TBP, regulatory
proteins interacting with the
subunit could modulate the ability of
TFIIA to associate with TBP. There is evidence that the Epstein-Barr
virus activator ZEBRA overcomes a mutation in the
subunit, which
decreases TBP binding and complex formation (29). Because the
subunit is upstream within the ternary complex (30, 31),
is
positioned to more easily interact with activators or possibly TAFs
that extend toward upstream bound activators.
Our understanding of how the transcription complex is assembled requires an understanding of each step, particularly the key regulatory ones. We have characterized the formation of a promoter-bound complex containing TBP and TFIIA. We will use this study as a starting point to perform parallel studies with TFIID and to examine the interactions of activators. It will be particularly interesting to determine how (and if) TAFs change the interaction of IIA with TBP. The ability of activators to both facilitate complex assembly and induce a conformational change in the DA complex could be studied. These future studies will benefit from using smaller proteolytic reagents, especially given the larger size of TFIID relative to TBP. Recent experiments by the labs of Meares, Heyduk, and Ebright and coworkers (47, 48, 49) have shown that hydroxyl-radicals can be an effective reagent for protease footprinting studies and will probably be a useful reagent for our studies.
We thank Tim Richmond for providing the coordinates of the TFIIA-containing ternary complex, Dimitar Nikolov and Steve Burley for providing the TFIIB-containing ternary complex, Richard Ebright and Arnie Berk for insightful discussions/comments on the manuscript, and Thai Nguyen for constructing some of the TFIIA deletion mutants.