©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Dimerization of the TATA Binding Protein (*)

Robert A. Coleman , Andrew K. P. Taggart , Lawrence R. Benjamin , B. Franklin Pugh (§)

From the (1) Center for Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 ) 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.


INTRODUCTION

The TATA binding protein (TBP)() 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 TBPTAF complex (SL1, TFIID, and TFIIIB, respectively). The complexity and low abundance of TBPTAF 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.

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 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 TBPTATA 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.

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 TBPTATA 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 TBPTATA interface appears to be predominantly hydrophobic and shows few sequence-specific contacts. The apparent equilibrium dissociation constant (K ) of the yeast TBPTATA complex has been reported to be 3 10M(10) and 2 10M(16, 17) , and for the human TBPTATA complex it has been reported to be 5 10M(18) .

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 TBPTATA 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.

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.


EXPERIMENTAL PROCEDURES

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 TBPTATA 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.

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 = 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 MeSO was added to a concentration of 0.1 mM (final concentration of MeSO in these reactions was 9%). Control reactions contained only MeSO. 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

On-line formulae not verified for accuracy

where S 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

On-line formulae not verified for accuracy

where S 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%.

The K for TBP dimerization was determined from the following equation using Kaleidagraph® software

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 ). 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.

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.


RESULTS

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 MeSO 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 TBPHis-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.

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.

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 of Dimerization

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 TBPTAF 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. TBPHis-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) 10M 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.


DISCUSSION

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= 4 10M 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.

Human TBP binds to the adenovirus major late TATA box (TATAAAAG) with an apparent K 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.

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 TBPTAF complexes such as TFIID exist as dimers is currently being investigated.

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.() 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.


FOOTNOTES

*
This research was supported by Grants GM47855 from the National Institutes of Health, and by the Searle Scholars Program/The Chicago Community Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 814-863-8252; Fax: 814-863-8595; E-mail: bfp2@psuvm.psu.edu.

The abbreviations used are: TBP, TATA binding protein; TAF, TBP-associated factor; pol, RNA polymerase; bp, base pair; poly(dG-dC), double stranded alternating polydeoxyguanylic-deoxycytidylic acid; BMH, 1,6-bismaleimidohexane; MeSO, dimethyl sulfoxide; NAB, nickel agarose binding.

L. R. Benjamin and B. F. Pugh, unpublished data.


ACKNOWLEDGEMENTS

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.


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