©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence for Functional Binding and Stable Sliding of the TATA Binding Protein on Nonspecific DNA (*)

Robert A. Coleman , 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 required at RNA polymerase I, II, and III promoters that either contain or lack a TATA box. In an effort to understand how TBP might function at such a wide variety of promoters, we have investigated the specific and nonspecific DNA binding properties of human TBP. We show that TBP has less than a 10-fold preference for binding a TATA box (TATAAAAG) than for an average nonspecific site. In contrast to TBP, which binds to the minor groove of DNA, major groove binding proteins typically display binding specificities in the range of 10. Once TBP is bound to DNA, whether it be a TATA box or nonspecific DNA, binding is quite stable with a t of dissociation in the range of 20-60 min for a 300-base pair DNA fragment. In this binding state, TBP appears to be capable of stable one-dimensional sliding along the DNA. Sequence-specific binding can be accounted for, in part, by different rates of sliding. Additional findings demonstrate that specific and nonspecific DNA impart upon TBP an enormous and equivalent degree of thermal stability, suggesting that the TBPDNA interface on nonspecific DNA is not radically different from that on TATA. Consistent with this notion, we find that nonspecifically bound TBP is competent in establishing pol II transcription complexes on DNA. Together, these finding provide a plausible mechanistic explanation for the ability of TBP to function at TATA-containing and TATA-less promoters.


INTRODUCTION

The TATA binding protein (TBP)() binds to the TATA box of eukaryotic promoters, assembles a transcription complex, and anchors it to DNA (1, 2, 3) . While promoters that contain TATA require it for proper function, many promoters lack TATA altogether and yet still require TBP (4, 5, 6) . For these TATA-less promoters, there appears to be little specific sequence requirement around the -30 region where TATA normally resides (7, 8) . TATA-less promoters, however, do rely on other cis elements such as Sp1-binding sites and initiator elements (5, 9) . Presumably, TBP is recruited through direct and indirect protein-protein interactions with other sequence-specific factors.

If TBP utilizes TATA to anchor transcription complexes to DNA, then what is the role of TBP at TATA-less promoters? Recent reviews have suggested dual functions for TBP (10, 11) . At TATA, TBP is bound tightly, while at TATA-less promoters, TBP is proposed to be either loosely bound to the DNA or bound indirectly via protein-protein interactions. If TBP is maintained in the transcription complex of TATA-less promoters primarily through protein-protein interactions, then is TBP only a linchpin between proteins of the preinitiation complex? Since many different proteins can link components within the preinitiation complex of all three nuclear RNA polymerases and yet not be conserved, the invariant requirement for TBP suggests that TBP does more than simply string proteins together. The universal requirement for TBP also argues for a common conserved function at TATA-containing and TATA-less pol I, II, and III promoters. If this common function is to anchor transcription complexes to DNA as it does at TATA, then how can TBP perform this function in the absence of TATA?

TBP binds to the minor groove of the TATA box (prototype sequence, TATAAAAG) (12, 13, 14, 15) . The minor groove of DNA has a paucity of functional groups with which to provide sequence specificity. The TBPTATA crystal structure indicates that monomeric TBP binds TATA primarily through hydrophobic and van der Waals' interactions with the bases and sugars (14, 15, 16) . These types of interactions could provide the bulk of the affinity of TBP for TATA, but in the absence of extensive hydrogen bonding, it is not evident how they might provide a high degree of specificity for TATA over nonspecific DNA. The equilibrium dissociation constant (K ) of the yeast TBPTATA complex has been reported to be 3 10M (17) and 2 10M(18, 19) , while the K for nonspecific (yeast genomic) DNA was 5 10M(19) , indicating a few thousand-fold lower affinity of yeast TBP for nonspecific DNA.

A lack of precise definition of the biochemical mechanism by which TBP binds DNA limits our understanding of the role of TBP in TATA-less (and TATA-containing) transcription. The DNA binding surface of TBP is sufficiently unique that paradigm DNA binding mechanisms, such as that for Escherichia coli RNA polymerase (20) , lac repressor (21) , and cro repressor (22) may be of limited relevance. We have set out to define the mechanistic basis of TATA and TATA-less transcription by exploring the dynamics of TBP interactions with TATA and nonspecific DNA. Using a nitrocellulose filter binding assay, we have measured the apparent equilibrium constant for TATA and nonspecific DNA binding and have found a very low degree of sequence specificity. The low specificity of TBP binding was particularly striking when examined by DNase I footprinting. DNA binding did not appear to be cooperative. We explored the mechanistic basis for this low specificity by measuring the dissociation kinetics of TBP bound to TATA and nonspecific regions of the footprinting probe. Surprisingly, the rate of dissociation from nonspecific DNA was very slow and not significantly different from the rate of dissociation from TATA. When multiple TBP molecules were present on the same footprinting probe, net dissociation from TATA did not proceed until adjacent nonspecifically bound TBP reached a critical minimum. One plausible interpretation of the data is that TBP stably translocates along the DNA prior to dissociating into the bulk solution. To probe whether the TBPDNA interface is similar to the TBPTATA interface, we performed thermal stability tests on specifically and nonspecifically bound TBP. We find that while TBP is extremely unstable when not bound to DNA, it appears to have a very high and equivalent degree of thermal stability on nonspecific DNA as it does on TATA. TBP bound nonspecifically to DNA also appears functional in assembling pol II basal transcription complexes. The data presented here lead us to conclude that TBP has the intrinsic capacity to bind quite stably to DNA and to establish a functional transcription complex, whether the underlying DNA is TATA or TATA-less.


EXPERIMENTAL PROCEDURES

Proteins, DNA, and Buffers

TBP was purified to homogeneity from recombinant E. coli cells using a three-step procedure involving polyethylenimine precipitation, phosphocellulose chromatography, and ammonium sulfate precipitation as described in the accompanying paper (33) and in Ref. 23. The final purified fraction is shown (see Fig. 1A). Different TBP preparations ranged from 50 to 70% active. TBP concentrations were 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). The concentration of active TBP was determined by titrations of TBP against known concentrations of TATA, which were well above the K for TATA binding. Sp1 was purified from HeLa cells infected with a recombinant vaccinia virus containing the Sp1 gene as described previously (6) . The 28-bp TATA oligonucleotide used in this study was chemically synthesized. The 300-bp GTI footprinting probe was generated by restricting the plasmid pS-GTI-141 with XbaI, end labeling with kinase, and cutting with PvuII. The probe was isolated using a polyacrylamide gel and quantitated spectroscopically using 1 A = 50 µg/ml. The STI probes, containing either wild-type or mutant TATA, were generated by polymerase chain reaction. For transcription assays, pS-GTI-141 was linearized with PvuII.


Figure 1: K for the TBPTATA complex. Panel A, silver-stained protein gel of purified human TBP. Panel B, determination of the fraction of TBP that is active for TATA binding. Increasing concentrations of TBP (total protein), as indicated, were incubated with 53 nMP-labeled 28-bp TATA oligonucleotide shown inside the figure. Bound complexes were separated from free by EMSA, as shown. This and additional data were quantitated and the fraction (F) of TATA bound determined as described under ``Experimental Procedures.'' F is plotted as a function of the molar ratio of total TBP to TATA. At F = 1.0, the reciprocal of x axis intercept represents the fraction of TBP that is active for TATA binding, assuming a binding stoichiometry of 1. This value multiplied by the total TBP concentration represents the concentration of active TBP. Panel C, binding reactions were set up as described under ``Experimental Procedures,'' using 0.05 nM 28-bp TATA oligonucleotide. Samples were analyzed either by EMSA () or by filter binding (). F and K were determined as described under ``Experimental Procedures.''



Binding Conditions

In addition to TBP and probe, binding reactions contained 20 mM Tris acetate (pH 7.5, 80% cation), 5% glycerol, 4 mM spermidine, 1 mM dithiothreitol, 75 mM potassium glutamate, 0.1 mM EDTA, 4 mM MgCl, 13.6 µM (nucleotides) poly(dG-dC), 0.01% Nonidet P-40, and 5 µg/ml bovine serum albumin in a volume of 50 µl.

Nitrocellulose Filter Binding Assay

This assay is used extensively for measuring protein-DNA complex formation and is described in more detail in Ref. 24. Briefly, reactions containing radiolabeled DNA were rapidly (5 s) filtered over nitrocellulose contained within a 96-well dot-blot apparatus (Bio-Rad) using a vacuum applied via a house water aspirator. The filters were washed and dried, and the radioactivity was quantitated using a Betascope (Betagen).

EMSA

Samples (10 µl) were loaded onto a native 4% polyacrylamide gel (prerun at 25 mA for 1-2 h at 4 °C) and electrophoresed under the ``prerunning'' conditions for 15 min. Gels were dried, and the radioactivity present was quantitated through a Betascope imaging system.

DNase I Footprinting

Reactions were diluted with 50 µl of DNase I solution (containing 0.012 units or 5 ng of DNase I (Worthington), 10 mM MgCl, and 5 mM CaCl), and the incubations continued for 30 s at 30 °C. Reactions were terminated with 3 M ammonium acetate and 125 µg/ml carrier tRNA. The DNA was precipitated with ethanol, subjected to electrophoresis on 8% polyacrylamide, 7 M urea gels, and the radioactivity was visualized by autoradiography.

Immunodepletions and Transcription

HeLa nuclear extracts were immunodepleted of TBP and TAFs with polyclonal anti-TBP antibodies bound to protein A-Sepharose as described previously (6, 25) . Transcription reactions were performed essentially as described previously (6, 25) and involve the incorporation of [- P]ATP into the body of the nascent RNA. RNAs were purified by phenol/chloroform extraction and electrophoresed on a 6% polyacrylamide, 7 M urea gel. The gel was dried, and the radioactivity was visualized by PhosphorImaging (Molecular Dynamics).

Calculations

For the filter binding and EMSA experiments where formation of a complex between the protein (in this case, TBP or Sp1) and radiolabeled DNA was being measured as a function of protein concentration, the fraction (F) of the DNA probe bound by the protein is described by Equation 1.

On-line formulae not verified for accuracy

When measured by EMSA, S is the radioactivity present in the region corresponding to the shifted protein-DNA complex at protein concentration x. If measured by filter binding, S is the amount of radioactivity retained on the filter paper at protein concentration x. S is the corresponding average radioactivity present when x = 0 (i.e. the absence of protein), and S is the average amount of radioactivity present when F becomes independent of x (i.e. when the reaction reaches the titration end point).

For DNase I footprinting experiments, where titration of a protein protects a specific region of the probe from cleavage by DNase I, the fractional occupancy of a particular region (F) was determined by Equation 2.

On-line formulae not verified for accuracy

S is the amount of radioactivity present in the protected region of the probe at protein concentration x. S and S are defined above.

For filter binding experiments where dissociation of a protein-DNA complex was being measured as a function of time, the equation used to describe the fraction of the protein-DNA complex remaining (F) is given by Equation 3.

On-line formulae not verified for accuracy

Each of the terms is defined above, except that the subscripts x, 0, and f denote units of time instead of protein concentration. With this equation, the initial conditions are set such that F = 1. The end point signal (S ) was generally found to be less than 5% of S.

For DNase I footprinting experiments where dissociation of TBP from a particular region (such as from TATA, the medium or low affinity regions) was being measured, the equation used to describe the fraction (F) of complex remaining is given by Equation 4.

On-line formulae not verified for accuracy

Apparent dissociation equilibrium constants, K, of the protein-DNA complexes were calculated from Equation 5 using Kaleidagraph software

On-line formulae not verified for accuracy

where P represents the uncomplexed protein concentration (in this case, TBP or Sp1). P is related to total active protein concentration (P ) by P = P- PD, where PD is the concentration of the protein-DNA complex. The apparent association equilibrium constant, K, is the reciprocal of K. Error determinations represent standard errors.

The apparent dissociation rate constant (k) was determined from Equation 6 using Kaleidagraph software.

On-line formulae not verified for accuracy


RESULTS

K for TATA

Recombinant human TBP was purified to homogeneity from E. coli cells (Fig. 1A). The fraction of TBP that was active for TATA binding was determined through EMSA by titrating known concentrations of TBP (determined by total amino acid analysis) against a known concentration of a TATA oligonucleotide (determined by UV spectrometry), which was well above the K for the TBPTATA complex (Fig. 1B). Assuming a stoichiometry of 1:1 for the TBPTATA complex, the TBP preparation shown in Fig. 1B appeared to be 55% active. Other preparations ranged between 50 and 70% active.

To determine the apparent K for the TBPTATA complex, TBP was titrated into reactions containing a radiolabeled 28-bp TATA oligonucleotide (5 10M). DNA binding was measured by two different assays, EMSA and nitrocellulose filter binding (Fig. 1C). Both assays yielded identical estimations of the apparent K, which was (4.6 ± 0.3) 10M. We consider this value provisional, since TBP dimerization (K= 4 10M) represents a competing reaction (see accompanying article (33) ). A more accurate determination of the K for dimerization will provide a better estimate of the true K for TATA binding. Since both EMSA and the nitrocellulose filter binding assays yielded identical results, which were no different than that reported for yeast TBP (17) , we assume that the filter binding assay provides a valid reflection of the binding properties of TBP.

K for Nonspecific DNA

A plasmid representing nonspecific DNA was cut into numerous different sized fragments and then radioactively end-labeled. The DNA fragments (each fragment present at 5 10M) were titrated with either TBP or Sp1 (for comparison) and allowed to reach equilibrium. After passage of the reactions over nitrocellulose, the bound DNA fragments were eluted and size separated by gel electrophoresis. The apparent association equilibrium constant (K ) for either TBP or Sp1 binding to each fragment was determined and plotted as a function of the number of nonspecific DNA binding sites on that fragment. As shown in Fig. 2 , a linear relationship between the apparent K and the number of nonspecific sites on each fragment was obtained. The slope of each line represents the affinity for each nonspecific site, which in terms of apparent K, was determined for TBP to be 3.4 10M and for Sp1 to be 3.0 10M. The scatter in the TBP data set showed precise reproduction upon multiple repeats and thus is most likely due to sequence-specific differences in intrinsic affinity for each fragment. As with our estimations for the K of the TBPTATA complex (Fig. 1C), the apparent K values for the nonspecific DNA fragments are considered estimates since TBP dimerization is a competing reaction. Since smaller nonspecific DNA fragments require higher amounts of TBP to achieve binding, the competitive effect of dimerization becomes more pronounced on these shorter fragments. As such, the true K for the smaller DNA fragments is probably higher than the apparent value. This deviation might be responsible for the negative y-intercept for the TBP plot. With this caveat, TBP appears to have <10 more affinity for the TATAAAAG sequence than for an average nonspecific site. For typical major groove binding proteins, the K for specific binding is in the range of 10M, and for nonspecific binding it is in the range of 10-10M, which represents a specificity factor of 10-10.


Figure 2: Determination of the K for nonspecific DNA binding. The plasmid pU-STI, which is essentially pUC118 with a few promoter elements, was restricted with HpaII, P-end labeled, and purified. The resulting population of 15 fragments ranged in size from 501 to 26 bp. Binding reactions were performed as described under ``Experimental Procedures'' with 5 10M of each fragment and over a range of TBP or Sp1 concentrations. After reactions were allowed to reach equilibrium (1 h, 30 °C), samples were filtered through nitrocellulose. The radioactive DNA fragments retained on the nitrocellulose were eluted with 0.1% SDS. The fragments were then size fractionated by electrophoresis on a native 8% polyacrylamide gel. The gels were dried, and the radioactivity in the following sized (bp) fragments was quantitated: 501/489 (combined), 431, 404, 369, 242, 190, 147, 131, 110, 67. A TATA box on this plasmid was present on a smaller fragment that was not considered in this analysis. The K values for each fragment were determined as described under ``Experimental Procedures.'' The reciprocal of the K (K) for the TBP-fragment complex () and the Sp1/fragment complex () were plotted as a function of the number of nonspecific binding sites/DNA fragment. Since a protein can nonspecifically bind double-stranded DNA in two directions, the number of nonspecific sites was taken as twice the number of base pairs. In addition, the number of sites that did not consist of at least 8 bp (due to their presence near the end of the DNA) was subtracted from total number of sites. The average K for a nonspecific site was taken as the slope of the line.



DNase I Footprint Analysis of Specific and Nonspecific Binding

DNase I footprinting allows the binding of TBP to a large number of sites to be compared with TATA within the confines of a single reaction. The 300-bp probe contains a TATA box and six 70% GC-rich Sp1 binding sites (Fig. 3A). Apart from the TATA box, none of the sequences were intentionally designed to contain TBP binding sites. As expected, 16 bp of DNA centered around TATA became protected at low TBP concentrations (Fig. 3B). As more TBP was added, a nonuniform protection pattern developed throughout the probe. A secondary medium affinity site bound TBP with only slightly lower affinity. At the highest level of TBP tested (34 nM), the entire probe became protected. Interestingly, based on calculations from the apparent K for nonspecific binding (Fig. 2), TBP should have a K for a 16-bp stretch of DNA (the length of a footprint), in the range of 10 nM. From visual inspection of Fig. 3B, this is the approximate TBP concentration where substantial nonspecific binding is observed. The correspondence of the filter binding and footprinting data suggest that the two assays are detecting the same phenomenon.


Figure 3: DNase I protection of specific and nonspecific DNA by TBP. Panel A, sequence of the upper strand of the footprinting probe used in this study (except panel D). The 300-bp probe was 5`-P-end labeled (indicated by the asterisk). Arrowheads correspond to DNase I cleavage sites that generated bands used to quantitate TBP binding to TATA and arbitrarily selected low and medium affinity sites. The underlined sequences correspond to the protected region encompassing the TATA box and the medium affinity site. The 8-bp TATA box is italicized. The 70-bp stretch preceding TATA contains the SV40 early promoter 21-bp repeats. These Sp1 sites are 70% GC-rich and would be expected to have very low affinity for TBP. Panel B, titration of TBP. Increasing nanomolar concentrations of active TBP, as indicated above each lane, were incubated with 0.5 nM footprinting probe at 30 °C. Probe indicates no TBP or DNase I added. The radioactivity in the regions corresponding to the groups of bars in panelB was quantitated using a Betascope imaging system. The same regions were also used in Figs. 4-7. The increased nonspecific protection observed at high TBP concentration is not due to artifactual DNA-independent inhibition of DNase I by the TBP preparation, inasmuch as addition of an excess quantity of competitor TATA oligonucleotide sequesters TBP and renders the footprinting probe completely susceptible to DNase I attack (see for example, Fig. 4). Panel C, sequence of the STI footprint probe. Panel D, titration of TBP on the STI probe containing a wild-type (TATAAAA) or mutated (TATGAAA) TATA, as indicated. The two probes are compositionally identical to each other except for the indicated point mutation, but compositionally different from the probe shown in panelA. Both probes were generated by polymerase chain reaction using the same radioactive primer, thereby ensuring that the probe concentrations were equivalent, inasmuch as equal amounts of radioactivity were present in both reactions. The region of the probe containing the wild-type or mutant TATA box is indicated by the graybox. DNA recovery markers are indicated by the arrows.



The binding of TBP throughout the probe at relatively low TBP concentrations might be a consequence of cooperative interactions with TATA-bound TBP, or the fortuitous presence of many strong TBP binding sites scattered throughout a sea of very weak sites. Indeed, yeast TBP has been shown to bind numerous different sites (26) . To address these issues, we constructed a completely different footprinting probe in which we inserted either a wild-type (TATAAAA) or a mutated (TATGAAA) TATA box (Fig. 3C). This AG transition had been previously reported to be one of the most deleterious single-point mutations of TATA (27) . When the two probes were titrated with TBP, the wild-type TATA box became protected at a lower TBP concentration than the mutant, as expected (Fig. 3D). As more TBP was added, the nonspecific regions uniformly became protected. The protection of the nonspecific DNA occurred at the same TBP concentration that protected the compositionally different probe, GTI, shown in panelsA and B. Therefore, TBP is unlikely to be binding to only a few high affinity sites fortuitously scattered throughout the probe. Most importantly, the mutated and wild-type TATA probes achieved equal levels of nonspecific protection at the same TBP concentration. Thus, the binding of TBP to nonspecific DNA did not appear to involve cooperativity with TATA-bound TBP. Indeed, as discussed above, the high degree of nonspecific protection at relatively low TBP concentration can be accounted for by the independently measured K for nonspecific DNA (see Fig. 2).

Mechanism of DNA Binding

The mechanistic basis for the apparently low specificity of TBP for TATA was investigated by examining the dissociation kinetics of TBP from TATA and nonspecific DNA. TBP was loaded onto the 300-bp footprinting probe so as to give complete DNase I protection (Fig. 4, lane0). The reactions were then challenged with a vast excess of unlabeled TATA oligonucleotide so as to initiate a net dissociation of TBP. Any TBP that dissociated from the probe into the bulk solution would become sequestered by the TATA oligonucleotide. After a period ranging from 0 to 180 min, the reactions were subjected to a 30-s DNase I treatment. As shown in Fig. 4, the low affinity region was the first to dissociate, followed by the medium affinity region and finally TATA. While the order of dissociation was not surprising, the high degree of stability on nonspecific sequences was unexpected. For instance, after 10 min, most of the low affinity region was still bound by TBP. The 70% GC-rich Sp1 binding sites and the medium affinity region exhibited significant levels of protection even after 30 min. Apparently, TBP has the intrinsic capacity to bind quite stably to nonspecific DNA sequences.


Figure 4: TBP dissociation from TATA and nonspecific DNA. 21 nM active TBP was preincubated with 0.5 nM footprinting probe for 40 min at 30 °C under the conditions described under ``Experimental Procedures.'' 1 µM unlabeled 28-bp TATA duplex oligonucleotide was then added, and incubations were continued at 30 °C for the time indicated above each lane (0-180 min). The reactions were then subjected to DNase I treatment, processed, and analyzed. NoTBP indicates reactions performed in the absence of TBP. indicates reactions in which the competitor TATA oligonucleotide was added before TBP. This latter reaction represents the final equilibrium level of dissociation (S). probe indicates no DNase I or TBP added.



In addition to qualitatively addressing the stability of TBP on TATA and nonspecific DNA, the data in Fig. 4provide clues on the kinetic mechanism governing TBP dissociation from TATA and nonspecific DNA. Paradigms for dissociation of sequence-specific major groove binding proteins from DNA have been well established. Proteins such as the lac and cro repressors dissociate from their cognate site into the bulk solution via two steps (21, 22) . Step one is a rate-limiting dissociation of the specifically bound protein to a nonspecific site located on the same DNA molecule. The second step involves the rapid dissociation of the nonspecifically bound protein into the bulk solution, where it can interact with other DNA molecules. According to this simple first-order dissociation scheme, the fraction (F) of initial protein-DNA complexes remaining over time (t) decays logarithmically according the equation ln F = -kt , where k is the apparent first-order dissociation rate constant for the entire dissociation process from a particular site. Since k is dominated by the rate at which the protein breaks sequence-specific contacts, a protein that binds to more than one site manifests different k values from each of the various sites (22, 28) .

If TBP behaves like a classical site-specific DNA binding protein, then we expect to observe a log-linear dissociation of TBP from TATA, the medium affinity region and the low affinity region as soon as excess TATA competitor is added. Moreover, since the sequence of the three regions differ, we expect k to also differ. Contrary to these predictions, when the natural log of the fractional occupancy of the low, medium, and TATA regions were plotted as a function of time, a quite different pattern emerged (Fig. 5A). While binding to the low affinity site decayed in a log-linear manner, the dissociation kinetics from the medium affinity site and TATA appeared biphasic. Dissociation from TATA and the medium affinity site appeared to wait until most of the TBP had dissociated from the lower affinity sites. Once net dissociation from TATA and the medium affinity site began, it proceeded in a log-linear manner (see also Fig. 7A), which did not appear to be significantly different from that of the low affinity site. Thus, dissociation of multiply-bound TBP from DNA reveals two unique phenomena: sequence-specific lags and apparent sequence-independent rates of dissociation.


Figure 5: Kinetic profile of dissociation from TATA and nonspecific DNA. Panel A, binding to regions corresponding to TATA (), the medium (), and the low () affinity regions of the dissociation time course shown in Fig. 4 were quantitated as described in Fig. 3 and under ``Experimental Procedures.'' lnF is the natural log of the fraction (F) of each region protected. F is plotted to the right of each graph. Values for k are plotted in Fig. 7B. Panel B, dissociation time course was performed as in Fig. 4, except that 3 nM active TBP was used (). Panel C, 21 nM active TBP was preincubated with 0.5 nM labeled 28-bp TATA oligonucleotide at 30 °C for 40 min under the conditions described under ``Experimental Procedures.'' To maintain a concentration of nonspecific DNA equivalent to that in panelA, reactions were supplemented with unlabeled pBR322. Net dissociation was initiated by the addition of 1 µM (50-fold molar excess over TBP) of unlabeled 28-bp TATA oligonucleotide. The fraction of TATA bound was determined through filter binding assays. Identical results were obtained with the gel shift assay. The dissociation rate constant, k, was determined to be 1.1 10 s (t = 100 min). This value agrees well with the value determined by Hoopes et al. (17) for yeast TBP using EMSA on a 110-bp TATA probe. However, by DNase I footprinting on the 300-bp probe k for TATA was determined to be 5.2 10 s (t = 22 min, panel B). At this point, we do not know the basis for this apparent difference.




Figure 7: Temperature and sequence independence of k. Panel A, binding to regions corresponding to TATA (), the medium (), and the low () affinity regions were quantitated as described in Fig. 3 and under ``Experimental Procedures.'' Panel B, the k values for TATA (), the medium (), and the low () affinity regions were determined from Figs. 4-7, and 0 °C data (not shown), and plotted as a function of temperature. The TATA box, being entirely composed of A and T might be partially or entirely melted at 70 °C, and thus might contribute to the few-fold enhanced dissociation at this temperature.



If TATA-bound TBP ``waits'' to dissociate until TBP has dissociated from other sites on the same DNA molecule, then two simple predictions can be made. 1) When the concentration of nonspecifically bound TBP is reduced, a corresponding reduction in the lag to TBP dissociation from TATA should be observed. Indeed, as shown in Fig. 5B, under lower TBP concentrations where only TATA and a portion of the medium affinity site were protected (3 nM TBP), the lag to dissociation was substantially reduced, while the rate of dissociation remained essentially unchanged. 2) In the absence of extensive flanking DNA sequences, i.e. on a shorter probe (28 bp P-labeled TATA oligonucleotide), even at a high TBP concentration, the lag to dissociation ought to be eliminated. As measured by filter binding (Fig. 5C), no lag to dissociation was observed on this 28-bp probe, and dissociation proceeded in a log-linear manner (although with somewhat slower kinetics). Thus, with longer DNA molecules, the presence of nonspecifically bound TBP seemed to delay the net dissociation of TBP from TATA. Apparently, net dissociation from TATA occurred only when nonspecifically bound TBP fell below a critical level. The only interpretation that we are aware of that can explain this data is that TBP constantly redistributes along the DNA prior to dissociation.

In Fig. 5A, dissociation of TBP from the low, medium, and TATA sites, once initiated, appeared to proceed at approximately the same rate (t = 20 min). If TBP rapidly translocates along DNA prior to dissociation, then the binding sites measured by DNase I footprinting are not necessarily the sites of dissociation into the bulk solution. TBP might instead dissociate into the bulk solution from a common point that, if rate-limiting, would yield sequence independent kinetics.

Temperature Effects on k

Thus far, the data suggest that TBP interacts with nonspecific DNA much in the same way as it interacts with TATA. To provide further evidence for this interpretation, we sought to identify a solution variable that might probe the TBPDNA interface. One such probe is temperature. TBP is known to be exquisitely temperature sensitive in its binding to TATA. Simply preincubating TBP at 47 °C for 15 min in the absence of DNA totally and irreversibly abolishes its DNA binding activity (29) . Since the intimate contact between a protein and DNA can stabilize a protein against unfolding (30, 31, 32) , temperature acts as a useful probe for comparing protein-DNA interfaces. If the nonspecific TBPDNA interface was more temperature sensitive than the TBPTATA interface, we would expect to observe rapid loss of TBP bound to nonspecific sites relative to TATA at elevated temperatures.

A dissociation time course equivalent to the one shown in Fig. 4 was performed at 55 and at 70 °C (Fig. 6). These temperatures inactivate unbound TBP in a matter of seconds.() Surprisingly, the pattern of dissociation at 55 °C was no different than at 30 °C, and only a few-fold faster at 70 °C. The presence of DNA, whether it be TATA or nonspecific, provided a dramatic and unprecedented increase (we estimate >1000-fold) in the thermostability of TBP. The magnitude of thermostability imparted by DNA provides a strong argument for the TBP interface with nonspecific DNA being qualitatively no different from its interaction with TATA.


Figure 6: Thermostable TBPDNA interactions. Dissociation reactions were set up exactly as described in the legend to Fig. 4. Equilibrium reactions at 30 °C were shifted to either 55 or 70 °C for 5 min to allow temperature equilibration. (95% temperature equilibration occurred within 2 min) Excess 28-bp TATA oligonucleotide was then added, which defines the zero time point. At the time (in min) indicated above each lane, the 55 °C reactions were footprinted with 0.0084 units of (3 ng) DNase I for 30 s at 55 °C as described under ``Experimental Procedures.'' At the indicated time, the 70 °C reactions were shifted to 30 °C and footprinted as described under ``Experimental Procedures.'' Control reactions, in which TBP was briefly incubated at the indicated temperature in the absence of DNA, then added to DNA at 30 °C showed no binding (data not shown). In these experiments, undigested probe ran as two bands, which can be seen in the lane marked probe as well as many of the other lanes. In native gels, the probe runs as a single band. We note that only full-length probe (which is more prevalent in reactions where TBP has not yet dissociated) behaves in this anomalous way. This anomaly is particular to the gel composition and does not affect interpretation of the data.



Quantitation of TBP dissociation from TATA, the medium, and the low affinity sites at 55 and 70 °C (Fig. 7A) revealed the same sequence-specific lag and the same sequence-independent rates of dissociation evident at 30 °C. Thus, even at these elevated temperatures, the same reaction mechanism seemed to hold. In Fig. 7B, k, for TATA, the medium, and the low affinity regions were plotted as function of temperature, ranging from 0 to 70 °C. k appeared surprisingly temperature-independent and sequence-independent over the extremely broad temperature range of 0-55 °C, and increased by less than a factor of 3 at 70 °C.

TBP-dependent Nonspecific Transcription

A functional test for nonspecific DNA binding is whether nonspecifically bound TBP is competent to assemble and initiate pol II transcription complexes. To examine both specific and nonspecific transcription, a plasmid containing 2.7 kilobase pairs of nonspecific sequences and the synthetic GTI pol II promoter (see Fig. 2A) was linearized 141 bp downstream of the transcriptional start site. We employed a run-off transcription assay using a HeLa nuclear extract, which was immunodepleted of nearly all endogenous TBP (and TBP-associated factors or TAFs) (6, 25) . As shown in Fig. 8 , lane1, pol II in this TBP-depleted nuclear extract has little intrinsic ability to transcribe DNA, either specifically or nonspecifically. The residual transcription observed in lane1 is not due to pol II since it is insensitive to 2 µg/ml -amanitin (see lane5). When pure TBP was titrated into the reaction, a 141-nucleotide transcript corresponding to TATA-mediated transcription of the GTI promoter was observed, as expected (lanes2-4). In addition, a large amount of nonspecific transcription was observed and increased as more TBP was added. Both specific and nonspecific transcription were sensitive to 2 µg/ml -amanitin, indicating that the transcripts were pol II-derived (lane5). As expected, both specific and nonspecific transcription were template-dependent (lane6). Although, the TBP used in this study was greater than 98% pure, it remains possible that the nonspecific pol II transcription was induced by a minor contaminant in the TBP preparation. To address this possibility, the TBP preparation was immunodepleted using affinity purified antibodies directed against either TBP or a control antigen (yeast BRF protein). As shown in lane7, the TBP preparation, which had been immunodepleted with anti-TBP antibodies, failed to support specific and nonspecific transcription, while the TBP preparation treated with the control antibodies fully supported both specific and nonspecific transcription (lane8). This finding confirms that the nonspecifically bound TBP is the species responsible for assembling functional pol II transcription complexes and is perhaps qualitatively acting no differently than if it were bound to TATA.


Figure 8: TBP-dependent nonspecific pol II transcription. The PvuII-cut plasmid pS-GTI-141 (200 ng) was incubated with active TBP as indicated above each lane and under the binding conditions described under ``Experimental Procedures.'' Reactions were incubated at 30 °C for 15 min in a volume of 15 µl. HeLa nuclear extracts (3 µl, 100 µg) were then added, and incubations at 30 °C were continued for 5 min. Transcription reactions were initiated with the addition of nucleotide triphosphates (including [-P]ATP). Reactions were terminated after 15 min, and the radiolabeled RNA was analyzed as described under ``Experimental Procedures.'' Since these extracts were also depleted of TAFs, and TAFs are required for basal pol III and pol I transcription, we do not expect and do not observe any TBP-dependent transcription arising from pol I and III (data not shown). It is well documented that TBP can assemble functional pol II transcription complexes in the absence of TAFs (6, 43, 44).




DISCUSSION

We have examined the equilibrium binding properties and dissociation rates of the human TBP with a TATA box and nonspecific DNA. We report the following observations. 1) TBP has a 740-fold higher affinity for the 8-bp TATAAAAG sequence (K= 0.5 nM) than for an average nonspecific 8-bp sequence (K= 370 nM). 2) TBP binding to DNA does not display cooperativity. 3) TBP dissociates into the bulk solution from TATA at approximately the same rate as it does when bound to flanking nonspecific DNA sequences. 4) When multiple TBP molecules are present on the same DNA molecule, net dissociation of TBP proceeds first from the lowest affinity sites and last from the highest affinity sites. 5) Both TATA and nonspecific DNA equivalently stabilize the DNA binding activity of TBP against thermal denaturation. 6) Nonspecifically bound TBP is active in assembling functional pol II transcription complexes. The most parsimonious interpretation, which can accommodate all of the data is that the TBPDNA interface is very similar at TATA and nonspecific sites, allowing TBP to function equivalently. The kinetics of dissociation also suggest that TBP binds DNA in an unusual multistep pathway, which includes stable one-dimensional diffusion along the DNA. Sequence-specific DNA binding appears to be achieved, in part, through sequence-specific rates of diffusion along the DNA.

Specific and Nonspecific DNA Binding

Sequence-specific and nonspecific DNA binding have been well characterized for major groove DNA binding proteins. Nonspecific interactions typically involve electrostatic interactions between positively charged residues of the protein and the negatively charged DNA phosphate backbone. Sequence-specific DNA binding involves additional interactions, most notably, hydrogen bonding with specific bases. The TBPTATA crystal structure provides a sharp contrast to these paradigms. TBP binds the TATA box though predominantly hydrophobic and van der Waals' contacts with the minor groove. Few hydrogen bonds are made. Moreover, the minor groove lacks the array of functional groups necessary for a protein to discriminate among bases with high specificity. Typical major groove binding proteins such as cro repressor have 10-fold higher affinity for a specific site than for an average nonspecific site (22) . For TBP, we obtain a specificity factor of less than 10. Possible mechanisms by which TBP achieves specificity are described further below.

The low specificity of TBP leads us to propose that the TBPDNA interface is very similar regardless of whether the sequence is TATAAAAG or nonspecific. A second observation in support of this proposal is that the rates of dissociation of TBP from TATA and flanking nonspecific sites into the bulk solution are nearly equivalent (t = 20-60 min). The slow dissociation rates reflect an intrinsically high stability on specific and nonspecific DNA. Major groove DNA binding proteins dissociate from nonspecific DNA 6 orders of magnitude faster (t estimated to be in the range of 1 µs) (22) . Third, the rates of dissociation into the bulk solution from TATA or nonspecific sites display very similar temperature insensitivity. In the absence of DNA, the TATA binding activity of TBP is rapidly inactivated at elevated temperatures. At 70 °C, unbound TBP inactivates within seconds. Yet when bound to TATA or nonspecific DNA, the DNA binding activity of TBP is very stable at these elevated temperatures. Either TBP is bound to nonspecific DNA in the same manner as it is bound to TATA or some other highly stable conformation exists on nonspecific DNA. There is no evidence to date that supports the latter possibility. Fourth, the biphasic kinetics of dissociation suggest that TBP bound to TATA is in dynamic equilibrium with TBP bound to nonspecific DNA, and maintains this equilibrium without dissociating into solution. The simplest interpretation of this kinetic data requires that TATA and nonspecific binding be qualitatively similar. Fifth, nonspecifically bound TBP appears to be active in assembling functional pol II transcription complexes. Finally, in all cases, crystal structure determinations of TBP with altered TATA boxes yields essentially the same structure as with TATAAAAG (45) . These combined results provide a strong argument for the interaction of TBP with TATA to be qualitatively no different than its interaction with nonspecific DNA.

Mechanism of DNA Binding

In Fig. 9, we propose a general mechanism for the interaction of TBP with DNA. The model proposes that TBP dimers (T ) must dissociate into monomers (T) prior to DNA binding. TBP monomers then rapidly preequilibrate with DNA (D) to form an unstable complex (TD) at an arbitrary site n. The unstable complex then isomerizes into a stable complex (TD). Once TBP is stably bound, it is free to randomly diffuse along the DNA to an adjacent site n + 1 (TD).


Figure 9: A mechanism for TBP association with DNA. T represents TBP dimers (discussed in more detail in the accompanying article (3)). T+ D represents a TBP monomer and DNA, respectively. TD corresponds to an unstable TBPDNA complex at arbitrary position n. TD represents a stable TBPDNA complex, and TD represents the same complex as TD but located one bp from n. Each of these reaction segments are described in more detail in the text.



Evidence for TBP dimerization as a competitive reaction with DNA binding is presented in the accompanying article (33) . The TBP dimer interface is proposed to overlap the concave DNA binding surface of TBP as envisioned in the dimeric crystal structure of the apo form of TBP (34, 35). We have determined an approximate K for dimerization to be 4 nM, which is sufficiently low so as to potentially dictate the kinetics of DNA binding. In addition, the kinetics of dimer to monomer conversion appear to be sufficiently slow (see Ref. 33) so as to account, in part, for the previously observed slow kinetics of TATA binding (17, 36) .

The rapid preequilibrium and isomerization segments of the binding reaction have been proposed previously by Hawley and co-workers (17) for TATA binding. The two-step model was based on the finding that k for TATA binding was measured to be approximately 1000-fold lower (10M s) than that expected of a one-step diffusion-limited reaction (10M s). However, no direct evidence for a two-step model was presented. In fact, at sufficiently high TBP concentrations, the rate of TBP binding became too fast to accurately measure (t < 1 min), thereby precluding detection of the proposed isomerization step (17) . These previously observed kinetics of TATA binding could also be explained totally or in part by a slow dimer to monomer conversion of TBP. Despite this caveat, TBP monomers might nevertheless proceed through a rapid preequilibrium prior to stable DNA binding. This assumption seems reasonable given that the TBPTATA crystal structure reveals a severely distorted DNA axis and widened minor groove upon TBP binding. The TATA box is unlikely to stably exist in such an extreme deformation in the absence of TBP. The proposed isomerization step might involve such a deformation. Indeed, prebending of the DNA has been found to enhance the binding of TBP (37) .

The evidence that TBP rapidly diffuses along the DNA relative to its dissociation into the bulk solution is derived from the observed biphasic kinetics of dissociation. When multiple TBP molecules are present on the footprinting probe, net dissociation of TBP from high affinity sites such as TATA proceed only when TBP bound to lower affinity sites reaches a critically low level. When flanking DNA sequences are removed, or if the initial TBP concentration is reduced, the lag to dissociation also diminishes. If TBP does not translocate along the DNA, then a log-linear decay of TBP bound to various sites would be expected immediately upon addition of excess competitor to initiate net dissociation from the probe. Since we do not observe cooperative binding between TBP molecules, we conclude that TBP dissociates into the bulk solution independent of other TBP molecules bound to the probe. The simplest interpretation of the lags prior to log-linear dissociation is that with every dissociation event, TBP constantly redistributes on the DNA according to its range of affinities for different sequences. For example, if TBP dissociates from TATA or any other site, then that site is rapidly refilled by TBP, which has translocated from a nearby lower affinity site. TBP is presumably dissociating from TATA or any other site at the same rate whether the probe contains a singly-bound TBP or is saturated with TBP, but net dissociation from TATA does not occur until the density of TBP on the DNA lattice has reached a sufficiently low level.

The binding mechanism outlined in Fig. 9has a number of implications. First, sequence-specific DNA binding would be achieved, in part, when k is different from k. In other words, the rate at which TBP slides will depend upon the underlying sequence. For example, TBP will slide off of the TATAAAAG sequence less often than TBP bound to the low affinity site described in this paper. Second, the rate-determining step in dissociation from TATA or a nonspecific site would be the proposed reverse isomerization step described by k. This mechanism infers that k will vary depending upon the sequence at n. Yet, the data presented here demonstrate that TBP dissociates from TATA and nonspecific DNA at essentially the same rate. This paradox can be reconciled by the fact that dissociation was examined from specific and nonspecific sites located on the same DNA molecule. If sliding (k ) is much faster than reverse isomerization (k ), then dissociation of TBP into the bulk solution from all sites, whether specific or nonspecific, will proceed from a common site on the DNA and thus appear to be sequence independent. Third, if k and k are sequence-dependent, then these steps provide additional means by which TBP discriminates among different sites.

Physiological Implications

The ability of TBP to bind stably and functionally to nonspecific DNA suggest that TBP, in the context of a TATA-less promoter, may be functioning no differently than TBP bound at a TATA-containing promoter. Perhaps the only difference between a TATA-containing and a TATA-less promoter is the extent to which the underlying sequence contributes to promoter specific binding. Presumably at TATA-less promoters, TBP must rely on other factors such as TAFs and transcriptional activators to achieve promoter specificity. Consistent with this, dTAF150 has been reported to bind the initiator element, which is an essential element at TATA-less promoters (38) , and activators such as Sp1, VP16, and NTF1 have been shown to bind TAFs (39, 40, 41, 42) .

If TBP binds quite stably to nonspecific DNA, then how is TBP prevented from becoming sequestered by the vast excess of nonspecific DNA present in the nucleus? Part of the explanation might lie in the sequence dependence of k and the potentially high value of k (see Fig. 9 ). In effect, TBP might be kinetically blocked from associating randomly with nonspecific DNA. At TATA-less promoters, activators might decrease k, which would increase the half-life of the unstable TD complex. Unwanted nonspecific DNA binding might also be minimized through TBP dimerization (see accompanying article (33) ). TBP dimers would decrease the concentration of TBP that is available for DNA binding (TD formation).


FOOTNOTES

*
This research was supported by grants from the National Institutes of Health, GM47855, and 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; pol, RNA polymerase; bp, base pair(s); poly(dG-dC), double stranded alternating polydeoxyguanylic-deoxycytidylic acid; EMSA, electrophoretic mobility shift assay; TAF, TBP-associated factor.

M. E. Portnoy and B. F. Pugh, unpublished data.


ACKNOWLEDGEMENTS

We thank T. Kodadek, B. T. Nixon, K. A. Johnson, and members of the Pugh lab for many helpful suggestions. We also thank H. M. Zuang for his efforts on the project.


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