From the
The TATA binding protein (TBP) is a central component of all
eukaryotic transcription machineries. The recruitment of TBP to the
promoter is slow and possibly rate limiting in transcription complex
assembly. In an effort to understand the nature of this potential
rate-limiting step, we have investigated the physical state of TBP
prior to DNA binding. By chemical cross-linking, gel filtration
chromatography, and protein affinity chromatography, we find that the
conserved carboxyl-terminal DNA binding domain of human TBP dimerizes
when not bound to DNA. The data completely support the proposed dimeric
structure of plant TBP, previously determined by x-ray crystallography.
TBP dimers are quite stable, having an approximate equilibrium
dissociation constant (K
The TATA binding protein (TBP)
TBP has a highly conserved 180 amino acid
carboxyl-terminal domain that binds DNA and interacts with a variety of
transcription
factors
(1, 2, 3, 8, 9) . The
amino-terminal portion is evolutionarily divergent both in length and
composition, and its function is unknown. The kinetics of TBP binding
to TATA are apparently slow (t
Biochemical and x-ray
crystallography studies on TBP reveal that it binds to the minor groove
of TATA
(11, 12, 13, 14, 15) .
The x-ray crystal structure of the TBP
In the absence of
DNA, the core DNA binding domain of plant TBP crystallized as a dimer,
with the dimer interface and the DNA binding domain
overlapping
(19, 20) . Since this dimeric state of TBP is
apparently incompatible with binding to the TATA box, it was generally
assumed not to reflect the physiological state of TBP, and might have
been a consequence of its close packing within the crystal. TBP has
been reported to form dimers, tetramers, and higher order multimers in
solution
(21, 22) . In addition, TBP has been reported to
bind TATA as a multimer
(21, 23) . Except for
dimerization in the absence of DNA, these observations are incompatible
with the dimeric TBP crystal structure as well as the monomeric
TBP
In
this paper, we examine the physical state of TBP before and after DNA
binding. By gel filtration, chemical cross-linking, and protein
affinity chromatography, we address whether TBP self-associates or
remains as a monomer in the absence of DNA. We then examine whether
TATA binding disrupts any potential self-association. The potential for
TBP to self associate is further characterized by determining its
approximate equilibrium constant, dissociation rate constant, and the
predominant force (electrostatic versus hydrophobic) that
drives self association. Finally, we discuss the implications that self
association has in controlling the binding of TBP to DNA and thus
transcription complex assembly.
The double-stranded
28 bp synthetic TATA oligonucleotide used in this study contained
adenovirus major late promoter sequences from -35 to -20.
The sequence of one strand is 5`-GGAATTCGGGCTATAAAAGGGGGATCCG-3`. The
DNA concentration of the double-stranded oligonucleotide was determined
spectroscopically, using 1 A
On-line formulae not verified for accuracy where S
On-line formulae not verified for accuracy where S
The K
On-line formulae not verified for accuracy where F is the fraction of total TBP (including His-180C)
that is dimeric. For simplicity, TBP and His-180C are considered
equivalent and their sum total denoted by
(T
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
Equations 5 and 6 combine and rearrange to yield the quadratic.
On-line formulae not verified for accuracy
Substituting the solution to Equation 7 into Equation 4 yields
Equation 3.
The NAB assay was also used to test whether DNA
binding and dimerization are mutually exclusive events. Reactions
containing TBP were incubated in the absence or presence of increasing
amounts of a TATA oligonucleotide. The mixture was then tested for its
ability to bind His-180C through the NAB assay. As shown in
Fig. 4C, when TBP was incubated with TATA, its ability
to bind His-180C was severely diminished. Equivalent levels of
poly(dG-dC) DNA had no effect on TBP binding to His-180C (data not
shown). These data confirm the interpretations of the cross-linking
results in Fig. 3that TBP dimerization and DNA binding are
competitive processes.
We present three different assays that together demonstrate
that TBP stably dimerizes when not bound to DNA. 1) By gel filtration,
the 38-kDa TBP migrates with a native size of 60-80 kDa. 2) In
the presence of the chemical cross-linker BMH, TBP rapidly cross-links
as a dimer. 3) A nickel-binding histidine-tagged version of TBP
(His-180C) captures an untagged TBP, allowing it to be specifically
retained on a nickel agarose resin in a 1:1 molar ratio. We envision
the dimer interface to encompass the concave DNA binding surface of TBP
as visualized in its x-ray crystal structure (see Fig. 8, and
Refs. 19 and 20). In agreement with the observed crystal structure of
the TBP dimer, we find that 1) dimerization occurs through the
conserved carboxyl-terminal domain of TBP, 2) the dimer interface is
hydrophobic, and 3) dimers must dissociate into monomers in order to
bind DNA. Dimerization is characterized by an approximate apparent
K
Human TBP binds to the adenovirus major late TATA box
(TATAAAAG) with an apparent K
The TBP concentration within a rat liver cell
nucleus has been reported to be in the range of 2-6
µM(22) . If this is true, then TBP within the
nucleus might exist predominantly as dimers when not complexed with
DNA. However, TBP is normally tightly complexed with numerous TAFs.
Whether TBP
We have also examined the stability
and kinetics of dimer subunit exchange. TBP dimers are stable to 1
M KCl, indicating that dimerization is not merely a
consequence of TBP being an electrostatically sticky protein. When the
kinetics of subunit exchange were examined, we found the exchange
process to be relatively slow, having a t of 11 min at 0
°C and 200 mM KCl. At high ionic strength (733
mM), the kinetics of exchange were slowed nearly 7-fold (t = 75 min) relative to that at 200 mM KCl. The high
salt stability of TBP dimers and the reduction in rate of subunit
exchange at higher ionic strength is in agreement with the crystal
structure prediction that TBP dimerization is driven by hydrophobic
forces
(19, 20) . At higher ionic strength, hydration of
exposed hydrophobic groups, which is predicted to be a necessary
intermediate in the subunit exchange process, becomes energetically
more unfavorable and thus slows the exchange kinetics. Consistent with
this, we find that low concentrations of the detergent Nonidet P-40
destabilize TBP dimers.
We thank A. Hoffmann and R. G. Roeder for the gift of
His-180C. We thank T. Kodadek, B. T. Nixon, K. A. Johnson, and members
of the Pugh lab for many helpful suggestions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) in the low
nanomolar range. The dimerization interface appears to be dominated by
hydrophobic forces, as predicted by the crystal structure. TBP dimers
do not bind DNA, but they must dissociate into monomers before stably
binding to the TATA box. Dissociation of TBP dimers appears to be
relatively slow, and as such has the potential to dictate the kinetics
of DNA binding.
(
)
is
required for the transcription of apparently all nuclear-encoded genes
(1-3). In eukaryotes, TBP is tightly associated with
TAFs
(1, 2, 4, 5) . Each of the three RNA
polymerases, pol I, II, and III, appear to function with a
compositionally distinct TBP
TAF complex (SL1, TFIID, and TFIIIB,
respectively). The complexity and low abundance of TBP
TAF
complexes unfortunately limits the detail in which they can be studied.
In vitro, TBP can assemble a functional pol II pre-initiation
complex in the absence of TAFs
(6, 7) . Inasmuch as TBP
contains many of the properties of TFIID and is easily purified in
large quantities, this protein is ideally suited to model many aspects
of TFIID function.
7 min for 3.5 nM
yeast TBP), which led to the suggestion that TBP binds TATA in a
two-step process analogous to the binding of Escherichia coli RNA polymerase to a promoter (10). Step one was proposed to
involve a rapid preequilibrium of TBP with the DNA, and step two is an
isomerization of the TBP
TATA complex into a stably associated
state. The slow kinetics, however, are also compatible with alternative
interpretations of the binding process, such as a change in the
physical state of TBP prior to DNA binding.
TATA complex shows a
splayed-open minor groove wrapped along the underside of TBP's
saddle-shaped DNA binding domain. Unlike most sequence-specific DNA
binding proteins, the TBP
TATA interface appears to be
predominantly hydrophobic and shows few sequence-specific contacts. The
apparent equilibrium dissociation constant
(K
) of the yeast TBP
TATA complex
has been reported to be 3
10
M(10) and 2
10
M(16, 17) , and for the human
TBP
TATA complex it has been reported to be 5
10
M(18) .
TATA co-crystal structure. Perhaps one explanation for the
observed multimerization of TBP is the tendency of proteins to
aggregate when not in their physiological milieu. In addition,
extensive chemical cross-linking may capture very transient
interactions that might not be physiologically relevant. With regard to
TBP multimers binding TATA, in the accompanying paper
(18) , we
demonstrate that TBP has relatively high intrinsic affinity for
nonspecific DNA. High affinity nonspecific DNA binding might be
misconstrued as binding by a multimeric form of TBP if binding
reactions are performed at appropriately high TBP concentrations.
Proteins
TBP was overexpressed in E. coli cells. Crude extracts (Fraction I) were precipitated with 0.25%
polyethylenimine; TBP remained in the supernatant. Polyethylenimine
supernatants (Fraction II) were passed over phosphocellulose (Whatman
P-11) at 0.35 M KCl, and step-eluted with H buffer (20
mM HEPES (pH 8), 10% glycerol, 2 mM MgCl,
0.05 mM EDTA, 1 mM dithiothreitol) containing 1
M KCl (Fraction III). TBP was precipitated with 0.11 g of
ammonium sulfate/ml of Fraction III. The precipitate was collected,
solubilized, and dialyzed against storage buffer (20 mM Tris
acetate (pH 8), 20% glycerol, 2 mM MgCl
, 0.1
mM EDTA, 1 mM dithiothreitol, 200 mM
potassium glutamate, and 0.1 mM phenylmethylsulfonyl fluoride)
to generate Fraction IV, which was used throughout this study. A
detailed purification protocol is available from the authors upon
request (or see Ref. 24). TBP prepared in this manner was >98% pure
(Fig. 1A) and was monodisperse as measured by S100
(Pharmacia) gel filtration chromatography. Different preparations of
TBP ranged from 50 to 70% active for TATA binding, in electrophoretic
mobility shift and filter binding assays where the TATA oligonucleotide
was present at concentrations at least 100-fold greater than the
K
of the TBP
TATA
complex
(18) . TBP (Fraction IV) concentration was determined by
total amino acid analysis and had an A
of 0.057
µg
ml, which can be referenced against bovine
serum albumin (Sigma, Fraction V, dry weight) having an
A
of 0.086 µg
ml or
-globulin (Pharmacia Biotech Inc., provided as dry weight
standard) having an A
of 0.043
µg
ml, in side-by-side reactions, all in 0.9
Bradford reagent (Bio-Rad).
Figure 1:
Purification of recombinant human
TBP. Panel A, silver-stained SDS-10% polyacrylamide gel of
human TBP purified from recombinant E. coli cells as described
under ``Experimental Procedures.'' Fraction I (lane2) represents 1 ppm of the crude E. coli extract, Fraction II (lane3) is 1 ppm of the
supernatant from the polyethylenimine precipitation. Fraction III
(lane5) represents 7 ppm of the 1 M KCl
step from phosphocellulose, while ft (lane4) represents 1 ppm of the phosphocellulose flow-through.
Fraction IV (lane6) represents 20 ppm of the
resolubilized and dialyzed ammonium sulfate precipitate. Panel
B, silver-stained SDS-10% polyacrylamide gel of an amino-terminal
truncated derivative of TBP termed His-180C, purified as described
under ``Experimental Procedures.'' Fraction A represents 1 ppm of the E. coli crude extract. Fraction
B represents 2 ppm of the solubilized ammonium sulfate
precipitate. Fraction C represents 60 ppm (lane 4)
and 600 ppm (lane 5) of the nickel-agarose eluate followed by
a Q-Sepharose flow-through. Protein molecular weight markers are shown
in lane1 of each
panel.
His-180C was overexpressed in
E. coli cells. Crude extracts (Fraction A) were precipitated
with 0.33 g of ammonium sulfate/ml of crude extract. The precipitate
was collected and solubilized in nickel wash buffer (20 mM
Tris-Cl (pH 8), 20% glycerol, 2 mM MgCl, 0.1
mM phenylmethylsulfonyl fluoride, 20 mM imidazole)
containing 200 mM KCl (Fraction B). Fraction B was mixed for 1
h at 4 °C with nitrilotriacetic acid-agarose (Qiagen) equilibrated
in nickel wash buffer containing 200 mM KCl. Resin was
collected by centrifugation at 2000 rpm for 5 min and washed 3 times
with nickel wash buffer containing 1 M KCl. Resin was added to
a 10-ml disposable column (Bio-Rad) and equilibrated with nickel wash
buffer containing 200 mM KCl, and His-180C was eluted using
nickel elution buffer (20 mM Tris-Cl (pH 8), 10% glycerol, 2
mM MgCl
, 200 mM KCl, 1 mM
dithiothreitol, 250 mM imidazole). His-180C was approximately
95% pure at this stage. The eluate was passed over Q-Sepharose
equilibrated in nickel wash buffer containing 200 mM KCl.
His-180C is present in the flow-through from this column (Fraction C).
Approximate His-180C protein concentration was determined by Bradford,
using TBP (described above) as a standard. Preparations of His-180C
were approximately 40% active for TATA binding.
= 50
µg/ml.
Gel Filtration Conditions
TBP (100 nM)
was incubated in 20 mM Tris acetate (pH 7.5, 80% cation), 4
mM MgCl, 4 mM spermidine, 1 mM
dithiothreitol, 0.1 mM EDTA, 5% glycerol, 75 mM
potassium glutamate, 1.4 µM poly(dG-dC), 0.01% Nonidet
P-40, and 5 µg/ml bovine serum albumin in a volume of 1 ml, for 60
min at 0 °C. The entire sample was loaded onto a Pharmacia S100 gel
filtration column (100 cm high, 16 mm diameter, 200 ml volume)
equilibrated with the same concentration of components as the binding
reaction (except that TBP and DNA were omitted), and developed at 20
ml/h. Fractions (2 ml) were collected and analyzed for TBP content by
Western blot using a dot-blot apparatus. Western blots were
photographed using type 665 Polaroid positive/negative film. The
intensity of the TBP signal was quantitated by laser densitometry.
Chemical Cross-linking
Reactions contained 75
nM recombinant TBP in 20 mM Tris acetate (pH 7.5, 80%
cation), 4 mM MgCl, 4 mM spermidine, 0.1
mM EDTA, 5% glycerol, 75 mM potassium glutamate, 345
µM poly(dG-dC), 0.01% Nonidet P-40, and the indicated
amounts of oligonucleotide or additional poly(dG-dC) in a volume of 10
µl. Reactions were incubated at 30 °C for 1 h. BMH (Pierce),
dissolved in Me
SO was added to a concentration of 0.1
mM (final concentration of Me
SO in these reactions
was 9%). Control reactions contained only Me
SO.
Cross-linking was allowed to proceed for 90 s at 23 °C before
quenching with the addition of 10 µl of 2
protein sample
buffer containing 1.42 M 2-mercaptoethanol. Samples were
electrophoresed on SDS 7.8% polyacrylamide gels followed by Western
blot analysis with anti-TBP antibodies.
Nickel Agarose Binding (NAB) Assay
Dimerization
reactions were performed in binding buffer (20 mM Tris-Cl, pH
8.0, 10% glycerol, 2 mM MgCl, and 100 mM
KCl) in a volume of 10 µl. Nitrilotriacetic acid-agarose (Qiagen)
was equilibrated with binding buffer. Samples (2 µl) were applied
to 5 µl of drained nitrilotriacetic acid-agarose (Qiagen) for 20 s
with constant agitation. The resin was then washed twice with 1.5 ml of
binding buffer. Proteins were eluted from the resins in Laemmli protein
sample buffer containing 0.5 M imidazole and electrophoresed
on an SDS 10% polyacrylamide gel. Protein bands were visualized by
silver staining. The intensity of each band was quantitated by laser
densitometry and normalized to the amount of His-180C recovered as
described below. All TBP band intensities were within the linear range
of silver staining.
Calculations
For NAB assay experiments, where
dimer formation is being measured, the fractional extent (F)
of dimerization is described by the following equation
is the signal corresponding
to the amount of complex formed at titration or time variable
x, S
is the signal present when x = 0 (i.e. the background present at the initial
conditions, which is typically less than 5% of S
),
and S
is the amount of complex formed when F becomes independent of x (i.e. when the
reactions reach the titration end point or equilibrium).
S
represent the band intensity signal on
silver-stained protein gels of resin-bound TBP. For the NAB assay,
recovery of the His-tagged protein (in this case, His-180C) is expected
to be constant, and so S
values are normalized
against the recovery of tagged protein by the following equation
is the raw
value for S
,
S
is the intensity of the
His-tagged 180C protein band, and S
is the average of
all the His-tagged 180C protein bands present in the titration or time
course. In general, S
varied by less than 10%.
for TBP dimerization was determined from the following equation
using Kaleidagraph® software
). Similarly, for Equations 4-7,
all dimeric TBP and His-180C species are denoted by (TT), and
all monomeric TBP and His-180C species by (T). Equation 3 was
derived from the following relationships.
Purity of Human TBP
Recombinant human TBP was
purified from E. coli cells in a three-step procedure
(Fig. 1A) and judged to be >98% pure by densitometry
of silver-stained protein gels. His-180C, which contains six contiguous
histidine residues at the amino terminus of a truncated version of TBP,
containing only the 180 carboxyl-terminal amino acids, was purified to
homogeneity by chromatography over nickel agarose and Q-Sepharose
(Fig. 1B). His-180C was judged to be >99% pure by
densitometry of silver-stained protein gels.
Gel Filtration
As a first step in examining the
ability of TBP to self associate, 100 nM TBP was subjected to
gel filtration chromatography under conditions optimized for DNA
binding (Fig. 2). TBP migrated with an apparent native molecular
mass in the range of 60-80 kDa, which is approximately twice its
monomeric size (38 kDa). No large molecular mass aggregates were
detected. If TBP self associates, it appears to form a complex
consistent with it being a dimer and not larger multimers under these
conditions.
Figure 2:
Gel filtration of TBP. TBP (100
nM) was loaded onto an S100 gel filtration column and
equilibrated with DNA binding buffer. The column was developed at 4
°C, and fractions were assayed for TBP by Western blot. Migration
of gel filtration molecular weight markers are indicated by the
arrows.
Chemical Cross-linking
Icard-Liepkalns
(21) demonstrated that BMH cross-links TBP into dimers and
tetramers. Significantly, no trimers were detected. We repeated these
cross-linking studies with our preparation of TBP, under conditions
optimized for DNA binding. Since we were interested in detecting stable
interactions and not random collisions, we drastically reduced the
concentration and incubation time of the cross-linker. TBP was
incubated at 30 °C with 0.1 mM BMH for 90 s, and then the
reactions were quenched with 2-mercaptoethanol. Proteins were
electrophoresed on an SDS-polyacrylamide gel and subjected to Western
blot analysis. In the absence of BMH, TBP migrated with a size of 44
kDa (Fig. 3, lane1). When TBP was briefly
incubated with BMH, a second species was observed migrating with a
molecular mass of approximately 90 kDa (lane2),
which is the size expected of a TBP dimer. We did not observe any
larger molecular mass species, indicating that under these conditions
TBP does not form stable higher order multimers that are susceptible to
BMH cross-linking. At high BMH concentrations (1 mM) and under
extended incubation periods (1 h), a small amount of higher order
complexes were observed (data not shown), as previously
reported
(21) . Since these higher order cross-linked structures
are generated only under conditions of extended time and cross-linker
concentration, they probably do not reflect a preassembled state of
TBP. We interpret these data to indicate that TBP can self associate
into dimers. Further evidence for dimerization is presented below.
Figure 3:
Chemical cross-linking of TBP. TBP (75
nM) was incubated for 1 h at 30 °C under the conditions
described under ``Experimental Procedures.'' In addition,
300, 150, 75, or 37.5 nM (molecules) TATA oligonucleotide
(lanes3-6, respectively) or a nonspecific
oligonucleotide (PSE (Ref. 26 and R. S. Carter, J.
Chicca, T. S. Fisher, and B. F. Pugh, submitted for publication),
lanes7-10, respectively) were included.
Lanes11 and 12 contained an additional 57
or 16.5 µM (nucleotides) poly(dG-dC), respectively. BMH
(0.1 mM) was then added to each reaction (except for the
control reaction shown in lane1 in which
Me
SO was added) for 90 s before quenching with 700
mM 2-mercaptoethanol. Samples were then electrophoresed on an
SDS 10% polyacrylamide gel, and probed by Western blot with anti-TBP
antibodies. Similar results were obtained with reactions performed in
the total absence of poly(dG-dC).
Since TBP crystallized as a dimer in the absence of
DNA
(19, 20) , but as a monomer in the presence of
DNA
(11, 12, 15) , we tested whether DNA binding
prevented dimerization. As shown in Fig. 3, lanes6-3, in the presence of increasing
concentrations of a TATA oligonucleotide, the level of cross-linked TBP
dimers decreased. In fact, there appeared to be an approximately 1:1
molar ratio of TBP to TATA in the disruption of the dimer. Equivalent
concentrations of a nonspecific oligonucleotide (lanes10-7) did not disrupt the TBP dimers. Additional
poly(dG-dC) (lanes11-12), which TBP has little
affinity for, also failed to disrupt the cross-linking. These
experiments were repeated with TBP incubated at 0 °C, and
essentially identical results were obtained (data not shown). These
results demonstrate that the high affinity binding of TBP to TATA
prevents dimer formation.
Nickel Agarose Binding Assay
To further
investigate and quantify the dimerization properties of TBP, we
developed a rapid NAB assay to measure both the equilibrium state and
dissociation rate of TBP dimers. A schematic of the NAB assay is shown
in Fig. 4A. A similar assay has been used previously to
qualitatively examine other protein-protein interactions
(25) .
In the NAB assay, a nickel-binding histidine tagged version of TBP
(His-180C) captures untagged TBP and retains it on nickel agarose in
the form of His-180C/TBP pseudo-homodimers. In practice, His-180C
homodimers and TBP homodimers are mixed together to allow subunit
exchange. After a defined period, samples are applied to a bed of
nickel agarose for 20 s. The resin was then rapidly washed to remove
unbound proteins. Proteins bound to the resin are eluted and analyzed
on silver-stained SDS-polyacrylamide gels. Since this type of assay has
not been previously used to measure the kinetics of protein-protein
interactions and since TBP dimerization kinetics also have not been
described previously, our initial goal was to provide a
semiquantitative analysis of the parameters governing TBP dimerization.
We emphasize that a rigorous assessment of each kinetic parameter is
beyond the scope of this study.
Figure 4:
Dimerization measured through the nickel
agarose binding (NAB) assay. Panel A, schematic of the NAB
assay. This assay utilizes a nickel-binding histidine-tagged version of
TBP (His-180C) to capture an untagged version of TBP on nickel-agarose
resin. His-180C homodimers (CC) are mixed with TBP homodimers
(TT). Subunit exchange will result in His-180CTBP
pseudo-homodimer (TC) formation. If equimolar amounts of C and
T are present, then dimers should form in the following ratio,
1CC:2CT:1TT. The reactions are then applied in batch to nickel-agarose
resin for 20 s. The resin is quickly washed in batch with binding
buffer to remove any unbound proteins (such as TT, which
represents 50% of the input T, if C and T are present in equimolar
ratios). Bound proteins (CC and TC) are eluted with imidazole buffer
and electrophoresed on an SDS-polyacrylamide gel. Since the kinetics of
TBP subunit exchange are sufficiently slow, the incubation time on the
resin represents only a small portion of the overall kinetic profile.
Panel B, TBP (4.2 µM, when present) and His-180C
(7.5 µM, when present) were incubated together (shown as
input in lane1) for 5 h at 0 °C under the
dimerization conditions described under ``Experimental
Procedures.'' In lane2, TBP was omitted. In
lane3, His-180C was omitted. Samples were applied to
nickel-agarose resin and washed extensively, and resin-bound proteins
were eluted and electrophoresed on an SDS 10% polyacrylamide gel and
stained with silver. Panel C, TBP (0.5 µM, of
which 0.3 µM is active for TATA binding) was incubated
with the indicated amount of TATA oligonucleotide for 50 min under the
conditions described under ``Experimental Procedures.'' The
reactions were then mixed with 7.5 µM His-180C immobilized
on nickel-agarose for 15 min at 30 °C. The resins were washed, and
the bound proteins were quantitated on silver-stained polyacrylamide
gels. The amount of TBP bound to His-180C in the absence of TATA was
arbitrarily set at 100.
To assess the validity of the NAB
assay and characterize the dimerization state of TBP, we first
performed equilibrium studies on TBPHis-180C pseudo-homodimers.
TBP and His-180C were incubated together and allowed to reach
equilibrium, and then the extent of pseudo-homodimerization was
determine using the NAB assay described in Fig. 4A. As
shown in Fig. 4B, lane2, His-180C was
rapidly and quantitatively removed from solution by the brief (20 s)
exposure to nickel agarose. Full-length TBP, which lacks the histidine
tag, did not bind the resin (lane3). However, when
His-180C and TBP were mixed together, the TBP that was present in
pseudo-homodimers was now retained (lane4). Similar
results were obtained using full-length His-TBP in place of His-180C
(data not shown). Binding was highly specific in that random E.
coli proteins did not copurify with His-180C on nickel agarose
(Fig. 1B, compare lanes3 and
4). The amount of TBP retained on the resin in the presence of
His-180C agreed well with the expected level of TBP
His-180C
pseudo-homodimer retention at these protein ratios and assuming a
dimeric structure for TBP (also see below). Moreover, these data
demonstrate that the conserved core DNA binding domain mediates
dimerization. While the amino-terminal domain is not essential for
dimerization, at this point we do not know whether it influences
dimerization.
Stoichiometry
To further verify that TBP is a
dimer, a fixed concentration of His-180C was titrated with excess TBP.
Once equilibrium was reached, the reactions were subjected to the NAB
assay. The amount of TBP retained on the resin (Fig. 5A,
lanes1-4) was normalized against His-180C
recovery, and quantitated against silver-stained TBP standards
(lanes5-7, and adjacentgraph). In Fig. 5B, the molar ratio of TBP to
His-180C retained on the resin was plotted against the input molar
ratio of TBP to His-180C. The dashedlines represent
the relationship expected of a tetramer and dimer, as indicated. In the
presence of increasing amounts of excess TBP, the binding stoichiometry
between TBP and His-180C more closely matched the binding pattern
expected for a dimer. The slight deviation might be due to loss of some
TBP during the wash step. When equivalent binding reactions were
compared at 0 and 30 °C, the ratio of TBP retained to His-180C
recovered was indistinguishable, indicating that there was no
difference in the stoichiometry of self association at 0 and 30 °C
(Fig. 5C).
Figure 5:
Determination of TBP dimerization
stoichiometry. Panel A, His-TBP (4 pmol, 1.7 µM)
was incubated with increasing input TBP concentrations (4, 8, 12, 16
µM in lanes1-4, respectively) at
0 °C and subjected to the NAB assay as described under
``Experimental Procedures.'' In lanes5-7, the indicated amount of TBP standard
(concentration determined by total amino acid analysis) was loaded, and
its staining intensity was plotted to the right (). The
amount of TBP retained on the resin in lanes1-4 was plotted on this standard curve, and the amount of TBP retained
was determined (
). The TBP signals were normalized to the amount
of His-180C recovered, which was approximately 80% in each case. (Input
His-180C standards are not shown.) Panel B, the normalized
amount of TBP retained in panels A and C (in pmol)
were divided by the input amount of His-180C to generate output
TBP/His-180C molar ratio, which was plotted as a function of input
TBP/His-180C molar ratio (
). The dashedlines represent theoretical curves based upon a dimer or tetramer
arrangement of TBP. Panel C, binding reactions were performed
exactly as described in Fig. 4B at either 0 or 30 °C and
subjected to the NAB assay. The input molar ratio was 0.6. The output
ratio of TBP and His-180C band intensities is presented below each lane.
K
Is TBP
dimerization an in vitro consequence of employing high
concentrations of TBP and thus of limited physiological relevance?
Ideally, one would determine the equilibrium constant for dimerization
and then compare its value with the equilibrium constants of its
competing reaction, DNA binding. In addition, TBP is generally
complexed with numerous other proteins such as TAFs, and so one would
also need to determine whether TBP of Dimerization
TAF complexes formed similar
dimeric structures at physiological concentrations. As a first step in
this direction, we sought to obtain an estimate of the equilibrium
dissociation constant (K
) for
dimerization. TBP and His-180C were allowed to reach equilibrium at 23
°C. TBP
His-180C pseudo-homodimers were then isolated via the
NAB assay, and the amount of TBP retained on the resin was quantified.
The fraction of total TBP and His-180C present as dimers (F)
was calculated as described under ``Experimental
Procedures,'' and the data were plotted in Fig. 6. The
fitted data yielded an apparent K
(± standard error) for dimerization of (4.0 ± 1.5)
10
M at 23 °C. Thus, it appears
that dimers can readily form at low nanomolar TBP concentrations. This
K
estimate is considered preliminary in
that the following untested assumptions were made: 1) the total TBP
concentration is equivalent to the TBP concentration that is active for
dimerization, and 2) the amino-terminal domain does not affect
dimerization. Direct assessment of these assumptions will provide a
firmer measure of the K
for TBP
dimerization.
Figure 6:
Determination of an approximate K for dimerization. TBP (10 µM) and His-180C (0.3
µM) were incubated for 30 min at 23 °C under standard
binding conditions, to allow maximum formation of TBPHis-180C
pseudo-homodimers. The mixture was then applied to nickel-agarose for 5
min. Aliquots of the resin slurry were diluted in binding buffer to
achieve the indicated final concentration of TBP. Incubation with
constant mixing was continued at 23 °C for 60 min. The resin was
then collected by centrifugation, washed, and eluted, and the protein
content was analyzed according to the NAB assay protocol (see
``Experimental Procedures''). The data were fit to Equation 3
described under ``Experimental
Procedures.''
Hydrophobic Dimer Interface
The TBP dimer
interface as envisioned in the crystallographic asymmetric unit cell is
hydrophobic (19, 20). If a similar dimer exists in solution, then we
expect it to be stable under conditions of high ionic strength. As the
ionic strength of a solution increases, it becomes increasing more
unfavorable to hydrate the exposed hydrophobic surfaces. As a
consequence, burying the hydrophobic residues through dimerization
becomes increasingly favorable. Fig. 7A shows that the
pseudo-homodimers formed between His-180C and TBP are maintained
through a 1-h incubation with 1 M KCl (the highest
concentration tested). The high salt stability is consistent with a
hydrophobic interaction and rules out dimerization as an artifactual
consequence of TBP being an electrostatically ``sticky''
protein. Note that this level of stringency was used to purify His-180C
from crude E. coli extracts in one step (see Fig. 1B and ``Experimental Procedures''), confirming that this
binding is highly specific.
Figure 7:
Evidence for a hydrophobic dimer
interface. Panel A, reactions were performed as described in
the legend to Fig. 4B and under ``Experimental
Procedures,'' except that the nickel-agarose resin containing the
bound proteins was incubated for 1 h at 0 °C with binding buffer
containing the indicate KCl concentration. Panels B and
C, time course of dimer exchange was performed as described in
Fig. 4, A and B, at the indicated KCl concentration. At
various time points (t), samples were removed and briefly
applied to nickel-agarose. In panel C, F denotes the
fractional extent to which pseudo-homodimer formation has reach
equilibrium. The data were fit to the equation: F = 1
- e, where
k
is the dissociation rate constant for the TBP
dimer.
To provide further evidence for a
hydrophobic interface and to examine the kinetics of dimer
dissociation, the rates of subunit exchange under high and low ionic
strength were compared. To measure the net rate of subunit exchange,
His-180C homodimers were mixed with TBP homodimers. At various times
after mixing, TBPHis-180C pseudo-homodimers were rapidly
separated from TBP homodimers using the NAB assay described in
Fig. 4A. Fig. 7B shows data from a time
course of TBP subunit exchange in the presence of 0.1 and 1.0
M KCl. In Fig. 7C, a more extensive time course
was performed at 200 and 733 mM KCl, and the quantitated data
presented. In both cases, the exchange kinetics were slower at the
higher salt concentration. At high ionic strength (733 mM
KCl), the exchange process slowed by nearly 7-fold relative to 200
mM KCl. The curves in Fig. 7C were fit to an
exponential decay (F = 1 -
e
), and the dissociation rate constants
(k
) for the TBP dimer were calculated to be 1.0
10
s
(t = 11 min) at 200 mM KCl and 1.6
10
s
(t = 75 min) at 733
mM KCl. This large reduction in exchange rate at high ionic
strength is expected of a dimer interaction driven by hydrophobicity. A
second property of TBP dimers, revealed by the data in Fig. 7,
B and C, is that dimer subunit exchange is
kinetically slow. The dissociation of TBP into monomers might be
sufficiently slow so as to control the kinetics of TATA binding.
= 4
10
M at 23 °C, and a slow rate of dissociation
(k
= 1.0
10
s
, t = 11 min at 200
mM KCl). The high propensity for TBP to dimerize and its slow
dissociation kinetics have the potential to significantly affect the
association of TBP to the TATA box, and thus transcription complex
assembly. In effect, the kinetics of TBP dimer dissociation might
dictate the kinetics of transcription initiation.
Figure 8:
A
model for TBP binding to TATA. In the absence of DNA, the concave DNA
binding domain of TBP self associates to form dimers, which cannot
accommodate additional monomers. The shadedarea represents the hydrophobic interface as viewed in the crystal
structure. Dimers slowly dissociate into monomers, which are then
capable of binding the TATA box.
Two studies have
provided biochemical evidence that TBP can self
associate
(21, 22) . However, these studies report that
TBP forms large aggregated complexes. Our findings with TBP indicate
that TBP predominantly self associates into dimers (see Fig. 2,
3, and 5). We detect no aggregation in the presence or absence of DNA.
The crystallographic model of TBP dimers provide no obvious mechanism
by which stable structures larger than dimers can form. In the report
by Icard-Liepkalns
(21) , higher order multimers were detected
predominantly by chemical cross-linking using prolonged incubation of
TBP in high concentrations of BMH cross-linker. Under such extreme
conditions, we also observe higher order structures (data not shown).
But under conditions of rapid cross-linking, we observe only dimers.
Under conditions of prolonged cross-linking where tetramers are
observed
(21) , no trimers were detected, indicating that
cross-linked dimers were most likely cross-linking to form tetramers.
We surmise that the higher order complexes result from cross-linking of
randomly colliding TBP dimers but do not represent a stable
interaction.
of 0.5
nM(18) . The apparent K
for nonspecific DNA binding is in the range of 400
nM(18) . We estimate that the upper limit for the
apparent K
of TBP dimerization is
approximately 4 nM. This number is considered tentative and is
expected to change once the amount of TBP that is active for
dimerization is accounted for. In addition, the amino-terminal domain,
which was absent in one of our dimer subunits, might affect
dimerization. The surprisingly high stability of TBP dimers has the
potential to competitively inhibit TATA box binding as well as
nonspecific DNA binding. Of practical concern is that reports of the
K
for TATA binding might be overestimates
of the true K
if a significant proportion
of TBP is dimeric.
TAF complexes such as TFIID exist as dimers is
currently being investigated.
(
)
The relatively slow
kinetics of dimer to monomer conversion has the potential to
significantly control the kinetics of TATA binding. It is plausible
that previous studies, which report slow association of TBP to TATA,
might have been observing a slow dimer to monomer conversion instead of
a slow direct association with DNA. Since TBP binding to TATA
represents an early step in transcription complex assembly, the
possibility exists that controlling the kinetics of dimer to monomer
conversion may, in effect, control transcription complex assembly and
initiation.
SO, dimethyl sulfoxide; NAB, nickel agarose binding.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.