DNA Bending is an Important Component of Site-specific Recognition by the TATA Binding Protein

JMB—MS 606 Cust. Ref. No. PEW 82/94

[SGML] J. Mol. Biol. (1995) 250, 434–446

DNA Bending is an Important Component of Site-specific Recognition by the TATA Binding Protein D. Barry Starr, Barbara C. Hoopes and Diane K. Hawley* Department of Chemistry and Institute of Molecular Biology University of Oregon Eugene, OR 97403, USA

*Corresponding author

We have used gel electrophoretic methods to analyze the extent, location and direction of the DNA bend induced by the TATA binding protein (TBP) upon binding to a consensus TATA box sequence. Our observations were consistent with the proposed models for the X-ray crystal structure of the TBP-TATA box complex. We have also measured the magnitude and direction of the bend induced by TBP upon binding a number of variant TATA box sequences for which we have measured TBP binding affinity. We found that the extent to which the DNA was bent in the complex differed among the various sequences and was correlated with the stability of the complex; that is, the greater the stability of the complex, the more the DNA appeared to be bent by TBP. This study provides the first evidence that the structure of the TBP-DNA complex may vary with different DNA sequences. In addition, we propose, based on our findings, that the energetics of bending contribute significantly to the overall binding affinity of TBP for different sequences. Keywords: DNA-protein interaction; TATA binding protein; eukaryotic transcription; DNA bending; DNA minor groove

Introduction The TATA binding protein (TBP) is a DNA-binding protein required by all three eukaryotic nuclear RNA polymerases for transcription initiation (Cormack & Struhl, 1992; Schulz et al., 1992; reviewed by Sharp, 1992; Hernandez, 1993). Recently, genes encoding close structural homologs of TBP have also been identified in archaebacteria, suggesting that this protein has an important role in transcription in those organisms as well (Marsh et al., 1994; Rowlands et al., 1994). The function of TBP has been studied most extensively in the context of the RNA polymerase (pol) II system, where TBP has been shown to recognize and bind a conserved promoter element known as the TATA box (Sawadogo & Roeder, 1985; Nakajima et al., 1988; Buratowski et al., 1988). The affinity of the TBP-TATA box interaction has been proposed to contribute to promoter strength both in vivo and in vitro (Myers et al., 1986; Wobbe & Struhl, 1990; Colgan & Manley, 1992), and subsequent assembly of the other general pol II transcription factors into a functional preinitiation complex is dependent upon this initial interaction at Present address: D. B. Starr, Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA 94143-0448, USA. B. C. Hoopes, Department of Biology, Colgate University, Hamilton, NY 13346, USA. 0022–2836/95/290434–13 $08.00/0

the TATA box (Nakajima et al., 1988; Davison et al., 1983; Fire et al., 1984; Buratowski et al., 1989). In vivo, TBP is tightly associated with other proteins (called TBP-associated factors, or TAFs) that appear to direct TBP to particular classes of promoters. For example, the pol II transcription factor TFIID consists of TBP and a number of other tightly associated polypeptides that are required in vitro for activated but not basal levels of transcription from TATA-containing promoters (Hoffmann et al., 1990; Kambadur et al., 1990; Peterson et al., 1990; Pugh & Tjian, 1990; Dynlacht et al., 1991; Tanese et al., 1991). For this class of promoters, the relative transcription activity observed in vitro for a variety of TATA box sequences is similar for TFIID and for isolated TBP, suggesting that the binding properties of TBP are not significantly modified by the other polypeptides in the complex (Wobbe & Struhl, 1990; Hoopes et al., unpublished results). Some pol II promoters and most pol I and pol III promoters do not contain a sequence that bears obvious similarity to the TATA box element, yet TBP is still required for transcription from those promoters (Pugh & Tjian, 1991; Zhou et al., 1992; Yoganathan et al., 1992; Comai et al., 1992; Lobo et al., 1992; White et al., 1992). In the case of TATA-less pol I and pol III promoters, TBP has been shown to function in association with different sets of polypeptides that bind together as a complex to the 7 1995 Academic Press Limited

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DNA without apparent sequence selectivity (Learned et al., 1986; Comai et al., 1992; Kassavetis et al., 1990, 1992; Taggart et al., 1992; White & Jackson, 1992). Although it is possible that in some contexts TBP does not directly contact the DNA or contacts the DNA differently than when bound to a TATA box, another possibility is that other proteins stabilize a weak interaction between TBP and non-canonical sequences (Comai et al., 1992; White & Jackson, 1992; Wiley et al., 1992; Zenzie-Gregory et al., 1993; Hernandez, 1993). Thus, the mechanism by which TBP targets a specific binding site and the extent to which this recognition is altered by other proteins with which TBP associates are important issues to resolve in understanding the role of TBP in initiation by all three eukaryotic RNA polymerases. The mode of interaction between TBP and its specific binding site is also interesting from a purely structural perspective because of several unusual features of the protein-DNA complexes (Klug, 1993). Among these features are the relative absence of specific protein-base hydrogen bonds, which appear to involve only the central two base-pairs of the TATA box, and the fact that all of the protein-base interactions occur entirely within the minor groove (Starr & Hawley, 1991; Lee et al., 1991; J. L. Kim et al., 1993; Y. Kim et al., 1993). Together, these findings raise the question of how TBP discriminates among different sequences. Although G-C substitutions at most positions within the TATA box are expected to interfere with TBP binding through steric interactions between the protein and the extracyclic 2-amino group on the guanine, the positiondependent discrimination among A-T and T-A pairs is harder to rationalize. The minor groove of B-form DNA does not appear to contain sufficient information to allow a protein to distinguish an A-T from a T-A base-pair, because of the nearly identical placement of the hydrogen bond acceptors, the N-3 of A and the C-2 oxygen atom of T (Seeman et al., 1976). However, indirect evidence based on the ability of different sequences to function as TATA boxes in the context of a pol II promoter suggested that TBP does not bind similarly to all A-T sequence combinations (Wobbe & Struhl, 1990). In an accompanying study (unpublished results), we directly measured the binding of TBP to a number of different A-T-rich sequences and showed that a single A-T to T-A transversion at one of several positions in the TATA box reduces the lifetime of the TBP-DNA complex by as much as tenfold to 50-fold. The demonstration that TBP does discriminate among A-T-rich sequences suggested that ‘‘direct readout’’ of the hydrogen-bonding groups on the nucleotide bases is supplemented by additional structural information that contributes significantly to differences in the binding free energy for complexes at different sites. The X-ray crystal structures of TBP-DNA complexes on two high-affinity TATA box sequences have revealed that the DNA in the complex is highly bent (J. L. Kim et al., 1993; Y. Kim et al., 1993). In this study, we have examined the possibility that the

magnitude and/or structure of the TBP-induced DNA deformation is different in lower-affinity complexes formed on other TATA box sequences. Based on the results of that analysis, presented here, we propose that the energetics of deforming the DNA in the complex contributes significantly to the selectivity of the interaction of TBP with different binding sites.

Results Circular permutation analysis of the TBP-induced bend on the consensus TATA box The first suggestion that TBP bends the DNA upon binding the TATA box was based on a technique called a circular permutation analysis (Horikoshi et al., 1992). In such an analysis, the DNA-binding site is placed at various positions in a set of circularly permuted but otherwise identical DNA fragments, and the electrophoretic mobilities of complexes containing those DNA fragments and the DNA-binding protein are compared (Wu & Crothers, 1984). The protein is generally inferred to bend the DNA if the DNA fragments in which the binding site is located near the center are more retarded in the gel than the fragments in which the site is located near one end. The location of the bend can be approximated by determining where the site must be positioned in the fragment to cause the greatest retardation of the mobility. Using this assay, Horikoshi and co-workers concluded that both human TBP (hTBP) and yeast TBP (yTBP) bend the DNA, although at different positions (Horikoshi et al., 1992). The magnitude and direction of the bend were not determined in that study, which used the consensus TATA box sequence of the adenovirus major late promoter (Ad MLP). As a first step in an analysis of TBP-induced bending on different TATA boxes, we repeated the circular permutation analysis with yeast and human TBP bound to the wild-type Ad MLP. We also tested a deleted version of yTBP containing only the highly conserved C-terminal domain (yTBP(D3-60)) to determine whether the N-terminal domain, which is highly variable among different species, had any effect upon the location and angle of the DNA bend induced by the full-length yeast protein. The set of permuted DNA fragments used in this analysis are shown in Figure 1A. The results of this experiment are shown in Figure 1B (lanes 1 to 5) for yTBP (D3-60) and (lanes 11 to 15) yTBP. As expected, DNA fragments in which the TATA box was nearer the center were more retarded in the gel than DNA fragments in which the site was nearer one end, consistent with the results reported by Horikoshi and co-workers. Comparison of the relative mobilities of the different complexes was consistent with the interpretation that yTBP(D3-60) bent the DNA at approximately the same location and to approximately the same magnitude as yTBP. This finding is consistent with the observation that most of the DNA-binding properties we and others have

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(a)

(b) Figure 1. Circular permutation analysis of yTBP and yTBP(D3-60) bound to the consensus TATA box. A, Representation of a portion of pUCBend(WT) with the relevant restriction sites. The various DNA fragments used in the circular permutation assays are shown. An arrowhead indicates the center of each fragment. Ad MLP sequences are represented by a filled rectangle and the TATA box by an open rectangle. The numbers indicate the distance, in base-pairs, from the center of the TATA box to the ends of the DNA fragments. B, The plasmid pUCBend(WT) was restricted with either BamHI (lanes 1, 6 and 11), Asp718 (lanes 2, 7 and 12), XmaI (lanes 3, 8 and 13), XhoI (lanes 4, 9 and 14) or BglII (lanes 5, 10 and 15) and labeled by filling in using Klenow fragment and either [a-32P]dATP (BglII, Asp718 and BamHI) or [a-32P]dCTP (XhoI and XmaI). Either 10 nM yTBP(D3-60) (lanes 1 to 5), buffer (lanes 6 to 10) or 10 nM yTBP (lanes 11 to 15) was incubated with each of the DNA fragments in a total volume of 15 ml. Binding conditions were as described in Materials and Methods. After 20 minutes at 30°C, 1.5 ml of 100 mg/ml poly[d(I-C)] was added; and, after 30 seconds, the reactions were loaded onto a native polyacrylamide gel containing 5 mM MgCl2 . Gel electrophoresis and subsequent manipulation of the gel were as described (Hoopes et al., 1992).

measured for yTBP and yTBP(D3-60) are very similar under the conditions used in this experiment (Kuddus & Schmidt, 1993; our unpublished results). However, Kuddus & Schmidt (1993) have reported that the N terminus appears to enhance bending in the complex with the wild-type Ad MLP, at odds with our observation. We do not know the source of this discrepancy. We have also used a circular permutation assay to examine the bend induced by the binding of hTBP (data not shown). In contrast to results reported by Horikoshi and co-workers, we noted no apparent difference in the behavior of yTBP and hTBP in this assay, suggesting that hTBP and yTBP both bend the

DNA at approximately the same location within the TATA box and to approximately the same angle (data not shown). The magnitude of the apparent bend was estimated to be about 95°, determined by comparison with the extent of retardation under the same electrophoresis conditions of a series of permuted DNA fragments containing an intrinsically bent DNA sequence (data not shown). This bend angle is in good agreement with that observed in the co-crystal structures (J. L. Kim et al., 1993; Y. Kim et al., 1993). The significance of this agreement is difficult to assess, however, as the determination of bend angles by this method is very dependent on the assumptions used to calculate it (Thompson & Landy, 1988; Zinkel & Crothers, 1990; Kerppola & Curran, 1991b). Nevertheless, we concluded that, by directly comparing the electrophoretic mobilities of TBP complexes formed on DNA fragments that differed only in the sequence at the TATA box, we could use the circular permutation assay to make valid comparisons of the relative magnitude of bending in complexes formed on different TATA box sequences.

Effects of TATA box mutations on the magnitude of the TBP-induced DNA bend One potential difficulty in analyzing the TBP-induced bends for the different TATA box sequences was that neither yTBP nor hTBP formed detectable gel shift complexes with most of the mutant TATA boxes, even though TBP could be shown by DNase I footprinting to form specific complexes with those sequences (unpublished results). However, we found that gel shift complexes could be detected for all of the sequences when we used the N-terminaldeleted protein yTBP(D3-60). The main reason for the ability to detect gel shift complexes with yTBP(D3-60) but not with the full-length protein appeared to be the greater stability of the yTBP(D3-60) complexes during gel electrophoresis (data not shown, and Hoopes et al., 1992). An enhanced stability during gel electrophoresis has been observed for complexes formed with a slightly different N-terminal deletion variant of yTBP (Kuddus & Schmidt, 1993). To obtain initial data on the magnitude of the TBP-induced bend on non-consensus TATA box sequences, we examined the gel mobility differences of TBP complexes located at the end or middle of the DNA fragment. Base-pair changes were introduced into the TATA box in the context of the plasmid used to generate the set of permuted restriction fragments; see Figure 1. In Figure 2, each DNA containing a mutant TATA box was restricted with BamHI or XhoI to place the TATA box near the end or in the middle of the DNA fragment, respectively. When the mutant TATA boxes were located near the end of the DNA fragment, all of the complexes with yTBP(D3-60) had similar mobilities in the gel (Figure 2, lanes 1 to 9). In contrast, when the TATA boxes were near the center of the fragment, we observed considerable

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Figure 2. Circular permutation analysis of yTBP(D3-60) bound to TATA box mutants. End-labeled BamHI (lanes 1 to 9) or XhoI (lanes 10 to 18) restriction fragments were isolated from the pUCBend plasmid series containing the wild-type, 31C, 30T, 29A, 29A27T, 28T, 28T27T, 27T and 25C TATA boxes, as described in the legend to Figure 1. The various DNA fragments were incubated with 10 nM yTBP(D3-60) for 20 minutes at 30°C and loaded onto a native polyacrylamide gel containing 5 mM MgCl2 . Gel electrophoresis and subsequent manipulation of the gel were as described (Hoopes et al., 1992).

variation in the mobilities of the complexes (lanes 10 to 18). This result indicated that the magnitude of the TBP-induced bend was significantly different in complexes with different TATA box sequences. The bend angles calculated from these data as for the wild-type Ad MLP TATA box complex are shown in Table 1. Effects of TATA box mutations on the location and direction of the TBP-induced DNA bend The circular permutation assay is not a definitive test of protein-induced bending for several reasons (Gartenberg et al., 1990). First, differences in electrophoretic migration due to changes in the location of a protein-binding site in the DNA fragment do not always indicate a protein-induced DNA bend. For example, although the yeast transcription factor GCN4 does not induce a significant bend in the DNA, it mimics that behavior Table 1. DNA-binding properties of yTBP(D3-60) with various TATA boxes Mutant

Sequencea

27T WT 28T27T 30T 28T 31C 29A27T 25C 29A

TATATAA TATAAAA TATTTAA TTTAAAA TATTAAA CATAAAA TAAATAA TATAAAC TAAAAAA

Bend angleb (deg.) 106 93 93 87 80 63 59 34 < 34

Lifetimec (min)

Relative transcriptiond

185 90–100 70 40 7–9 4 1.5 8 1

0.73 1.0 0.72 0.68 0.35 0.41 0.15 0.24 0.11

a The various TATA boxes are within the context of the Ad MLP (see Materials and Methods). b Bend angles were calculated as described in Materials and Methods. c Lifetime measurements were obtained by either gel mobility shift analysis alone (WT, 27T, 28T27T and 30T), DNase I footprinting alone (29A and 29A27T) or both (31C, 28T and 25C). d Transcription values were obtained using 24 nM yTBP (D3-60).

in a circular permutation assay, possibly because of its elongated shape (Gartenberg et al., 1990). Thus, it was possible, though not likely, that some property of the TBP-DNA complexes other than bending contributed to the differences in electrophoretic mobilities observed for the different TATA boxes in the experiment illustrated by Figure 2. Second, a circular permutation assay does not provide any information that can be used to deduce the direction of the bend, whereas an assessment of whether the bend appears significantly altered in structure as well as magnitude is important to our analysis. A complementary assay developed in several laboratories provides additional information that can be used to verify that the DNA is directionally bent and to calculate the global geometry of the bend (Zinkel & Crothers, 1987; Salvo & Grindley, 1987). This technique involves insertion of a sequence of DNA that contains an intrinsic bend of known magnitude and direction into the same DNA fragment as the binding site of the protein of interest. The ability of the intrinsic bend to amplify or cancel the effects of the protein-induced bend are then assessed as the distance between the two bending loci is varied to place them on the same or opposite sides of the DNA helix. This method, called phasing analysis, provides an estimate of the magnitude and direction of the protein-induced bend. We placed a sequence that is intrinsically bent toward the minor groove at six different positions relative to the TATA box (Figure 3A). We restricted these DNAs so that the TATA box was in the center of the fragment and determined the mobility in a gel shift assay for complexes containing yTBP (Figure 3B, lanes 1 to 6), yTBP(D3-60) (lanes 7 to 12), and hTBP (lanes 13 to 17). The relative mobilities of the complexes are plotted versus distance between the sites in Figure 3C. As expected if TBP were truly inducing a bend in the DNA, the mobilities of the complexes varied in a periodic fashion as the distance of the TATA box from the intrinsic bend varied. All three TBP proteins showed the same pattern of relative mobilities in this assay, demonstrating that the TBP-induced bend was similar in magnitude, location and direction for each of the proteins. In the slowest-migrating complexes observed with all three forms of TBP, the intrinsic bend was located 47 base-pairs from the base-pair within the TATA box that corresponds to position − 28 (TATAAAA). In the fastest-migrating complexes, the intrinsic bend was located 52 base-pairs from position − 28. Assuming a helical repeat of 10.5 base-pairs per turn, this finding indicated that the intrinsic bend contributed most to the altered mobility induced by TBP when located on the opposite side of the helix from the TATA box (a separation of the two sites by 4.5 helical turns) and tended to cancel the TBP-induced bend when on the same side of the DNA helix (a separation of 5.0 helical turns). These data, then, suggested that the bend induced by TBP has the opposite direction from the intrinsic bend, and is directed toward the major groove at the center of the TATA box. Assuming that

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A

Figure 3. Phasing analysis of yTBP, yTBP(D3-60) and hTBP bound to the consensus TATA box. A, The relevant sequences of the various phasing plasmids are shown with the distance between the center of the intrinsic bend and the TATA box indicated. The underlined sequences differ among the various plasmids. B, The plasmids pUCBend + 0WT and pBWT +2, +3, +5, +8 and +12 were restricted with XhoI and labeled by filling in using Klenow fragment and [a-32P]dCTP. Either 10 nM yTBP (lanes 1 to 6), 10 nM yTBP(D3-60) (lanes 7 to 12) or 0.2 ml of hTBP (lanes 13 to 17) was incubated with each of the DNA fragments as described in the legend to Figure 1B. After 20 minutes at 30°C, 1.5 ml of 100 mg/ml poly[d(IC)] was added to reactions containing yTBP or yTBP(D3-60), which were then incubated for an additional minute at 30°C and loaded onto a native polyacrylamide gel. Reactions containing hTBP were incubated for 45 minutes at 30°; 1.5 ml of 0.22% Sarkosyl was then added and, after an additional three minutes at 30°C, the reactions were loaded onto the gel. Gel electrophoresis and subsequent manipulation of the gel were as described (Hoopes et al., 1992). The differences observed in electrophoretic mobilities for the unbound DNA fragments are consistent with the expectation that the intrinsic bend of the A-tract of the consensus TATA box collaborates with the clustered A-tract when the two are separated by an integral number of helical turns and offsets the overall bending when the two sites are spaced at other distances. C, The relative mobilities (distance migrated by the bound DNA/distance migrated by the free DNA) of the various TBP-TATA box complexes are plotted versus the distance (in base-pairs) between the center of the A-tracts and the TATA box.

the intrinsic bend induced by the A-tracts is 54° (Koo et al., 1990), the magnitude of the TBP-induced bend was 90 to 100°, consistent with the bend angle determined by the circular permutation assay. Both the direction and magnitude of the bend were

consistent with the structure of the TBP-TATA box complex determined by X-ray crystallography (J. L. Kim et al., 1993; Y. Kim et al., 1993). The results of the circular permutation and phasing analyses of yTBP complexes on wild-type

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Ad MLP placed the center of the bend at or near the middle of the TATA box, as expected. Next, we considered the possibility that some of the mutations might appear to change the locus of the bend, providing clues to the nature of the altered interaction with the DNA. In order to address this possibility, the intrinsically bent sequences used in the experiment illustrated by Figure 3 were placed upstream of each of the TATA box mutations, and the resulting DNA fragments were complexed with yTBP(D3-60) and analyzed using the phasing analysis described above. Figure 4 shows the relative mobility of each complex plotted versus the location of the intrinsic DNA bend for all of the TATA box variants. We found that yTBP(D3-60) appeared to bend all of these sequences at the same position and in the same direction but to either a greater or lesser extent. This conclusion is strongest for mutations with less severe effects on TBP-induced bending, such as 30T (TTTAAAA), 27T (TATATAA), 28T27T (TATTTAA) and 28T (TATTAAA), for which the magnitude of the bend angle was relatively large. For the mutations with more severe effects, such as 29A (TAAAAAA) or 25C (TATAAAC), the differences in mobility among the collection of DNA fragments were so slight that a shift of bend locus by one or two base-pairs would probably not have been detected. Nevertheless, the results in Figure 4 are consistent with the general conclusion that the TATA box mutations affected primarily the extent and not the overall direction or locus of the TBP-induced DNA bend. This finding is somewhat surprising in light of the X-ray crystal structures, which have shown that the DNA is not simply bent in the center of the TATA box, as might be predicted from our results, but instead is kinked in two places and unwound in between (see Discussion). Correlation of the magnitude of the bend with complex stability and transcription activity One of the motivations for measuring the bend angle observed with different TATA box sequences was so that we could determine whether the relative magnitudes of the bend correlated with the relative stabilities of the different complexes. To make this comparison, we determined the lifetimes of yTBP(D3-60) bound to each of the different TATA box sequences using DNase I footprinting and/or gel mobility shift assays, as we have done for the full-length yeast TBP bound to the consensus TATA box (Hoopes et al., 1992). We have found that, for full-length yTBP bound to different DNA sequences, the lifetimes of the complexes correlate well with their affinities (unpublished results). The results of this analysis are listed in Table 1. In all cases, the lifetime measured for yTBP(D3-60) was very similar to that determined for full-length yTBP on the same TATA box under the same solution conditions. We also measured the ability of yTBP(D3-60) to support basal transcription from each of the TATA boxes

(Table 1). In this assay, as in the direct measurements of the binding properties, the relative ability of yTBP(D3-60) to interact functionally with the different TATA box sequences was similar to that observed for the full-length protein (unpublished results). The data in Table 1 indicate that the magnitude of the bend and both the stability of the complex and the amount of basal transcription generally show a positive correlation. That is, the complexes with longer lifetimes tended to appear more bent and to promote a higher level of transcription. For example, the sequence TATTTAA, which forms a complex with a lifetime similar to the consensus TATA box, also is bent in the complex to about the same extent. The other mutant TATA boxes are ranked in the Table in order of the magnitude of the bend, and the complex lifetimes can be seen to follow the same trend, decreasing as the bend angle decreases. TATAAAC was the only obvious exception to this trend, having a longer lifetime than other TATA boxes that were bent to a similar, or even somewhat greater, extent. Strikingly, the one sequence that showed a greater degree of bending in the complex than the wild-type TATA box, TATATAA, also was the only complex with a longer lifetime. With the exception of this sequence, CATAAAA, and TATAAAC, there was also a general correlation between the extent of TBP-induced bend and the level of basal transcription. The amount of transcription observed for TATATAA was reduced relative to the consensus TATA box, possibly reflecting a TBP-DNA complex in which the DNA was bent more than is optimal for formation of the initiation complex. Alternatively, some of the transcription complexes may have contained TBP bound in the wrong orientation with respect to the start site, or the complex may have been so stable that the eventual release of the polymerase from the promoter was somewhat inhibited. In contrast, the TATA box with a T to C change at −31 (CATAAAA) or an A to C change at −25 (TATAAAC) promoted more transcription than might have been expected if the overall TBP-induced bend angle were the sole determinant of the relative ability of a particular TATA box sequence to function in transcription initiation.

Discussion In this study, we have examined the relationship between the affinity with which TBP binds to a collection of TATA box sequences and the apparent bend angle of the DNA in the various complexes. We found that base-pair changes within the TATA box affected the extent to which the DNA appeared to be bent in the TBP-DNA complexes observed by native gel electrophoresis, providing the first evidence that the predominant solution structure of the TBP-DNA complex can vary with different DNA sequences. Furthermore, we observed a correlation between the degree of bend and the stability of the complex in solution such that complexes with shorter lifetimes appeared to have decreased bend angles. Although

JMB—MS 606 440 most of the TBP-DNA complexes dissociated more rapidly than the complex containing the consensus TATA box (TATAAAA), one TATA box in our study (TATATAA) formed a complex with a longer lifetime

DNA Bending in TBP Recognition

than the consensus TATA box complex. This was also the only sequence to form a complex that appeared to be bent to a greater extent than the consensus sequence. One sequence, TATTTAA, bound TBP with

Figure 4. Phasing analysis of yTBP (D3-60) bound to mutant TATA boxes. End-labeled XhoI fragments were isolated from the pB + 0, +3, +5, and +8 series of plasmids containing the wild-type, 31C, 30T, 29A, 29A27T, 28T, 28T27T, 27T and 25C TATA boxes. The reactions and gel conditions were as described in the legend to Figure 3, except that poly[d(I-C)] was not added. The relative mobilities (distance migrated by the bound DNA/distance migrated by the free DNA) of the various yTBP(D3-60)-TATA box complexes are plotted versus the distance, in base-pairs, between the TATA box and the center of the phased A-tracts for the following TATA box sequences: A, TATTAAA, TTTAAAA and TATATAA; B, CATAAAA, TATTTAA and TAAATAA; and C, TAAAAAA and TATAAAC. The bend angles calculated from these data as described in Materials and Methods were: TATATAA, 91°; TATTTAA, 91°; TTTAAAA, 89°; TATTAAA, 69°; CATAAAA, 62°; TAAATAA, 37°; TATAAAC, 27°; and TAAAAAA, 25°.

JMB—MS 606 DNA Bending in TBP Recognition

about the same affinity as the consensus TATA box and was also bent to a similar extent. In the proposed X-ray crystal structures of the TBP-DNA complex (J. L. Kim et al., 1993; Y. Kim et al., 1993; Kim & Burley, 1994), only the two central base-pairs of the TATA box interact with the protein via hydrogen bonds to amino acid side-chains. These interactions, as well as a number of van der Waals contacts between the bases and protein, occur entirely within the minor groove of the DNA helix. In the complex, the TATA box is unwound and smoothly bent as a result of the widening and flattening of the minor groove, which forms a water-excluding interface with the protein. The crystal structures also revealed an additional contribution to the TBP-induced bend caused by two sharp kinks in the DNA backbone, one at each end of the TATA box, where phenylalanine residues partially intercalate between the base-pairs. It was not possible to predict, a priori, how or even whether the overall structure of the complex, as measured by a change in DNA bending, would be altered when TBP was bound in complexes of different affinities. The structure of each complex depends on how the favorable and unfavorable contributions to the free energy of complex formation balance to achieve the lowest free energy state. A base-pair change within the TATA box could reduce the affinity of the complex either by changing the protein-DNA contacts (removing a favorable interaction or introducing an unfavorable interaction), or by increasing the energetic costs of deforming the DNA (or, possibly, the protein) in the complex, or both. We cannot assign values to any of the positive or negative free energy terms in the overall free energy equation describing these complexes nor can we predict the effects of altering one property of the structure (e.g. a protein-DNA contact) on other structural features of the complex (e.g. the DNA or protein conformation). For example, whereas bending the DNA less would be energetically less costly, the protein-DNA contacts and, possibly, the protein conformation, would have to adjust to accommodate the less bent structure. These changes could, in themselves, result in further loss of binding free energy. In fact, our finding that the high-affinity complexes were more bent than low-affinity complexes indicated that, at least for some sequences, the increased bend angle significantly improves the quality and quantity of protein-DNA contacts. It is likely that a reduction in favorable proteinDNA contacts contributed to at least some of the observed reduction in affinity among the various complexes. However, several observations suggested that differences in the energetics of bending the DNA also contributed to the structures and affinities of complexes formed on non-consensus TATA sequences. First, most of the sequence changes that decreased the complex affinity in this study were A·T to T·A base changes, which would be expected to cause minimal disturbance of the minor groove surface with which TBP interacts. Indeed, several of these A·T to T·A changes were more deleterious to

441 binding than the introduction of a G·C base-pair, which would be expected to interfere with the tight contact between the protein and the minor groove surface because of the extracyclic amino group of the guanine in the minor groove. Second, we found that some base changes that had no or little effect on affinity or bend angle in one sequence context reduced the affinity and bend angle in another context (e.g. introduction of a T·A base-pair at position −28 was deleterious in the sequence TATTAAA but not when combined with another T at −27 in the sequence TATTTAA). This observation is most readily reconciled with a model that allows for the possibility that nearest-neighbor effects between DNA bases contribute in a significant way to the energy costs of DNA bending and, thereby, to the determination of the lowest free energy structure. There are several possible reasons why particular DNA sequences could be more difficult than others to deform to the structure observed in the high-affinity TBP-DNA complexes. For some DNA sequences, the lower-affinity binding could be associated with the necessity to distort the DNA in a manner that opposes a preferred solution structure. Several lines of evidence suggest that some of the TATA box sequences in our study would be expected to significantly occupy a solution structure featuring a narrowed minor groove relative to canonical B-form DNA. In particular, a series of four or five consecutive A bases, such as occurs in the 3' half of the consensus TATA box, would be expected to be compressed toward the minor groove (Burkhoff & Tullius, 1987; Zinkel & Crothers, 1987; Salvo & Grindley, 1987). Consistent with that expectation, the consensus TATA box and some of the derivatives we have studied are relatively resistant to cleavage by DNase I (unpublished results), which binds and cleaves through the minor groove (Lahm & Suck, 1991). The tendency of A-tracts to bend toward the minor groove could help explain both the stronger interaction of TBP with TATATAA, where the T at position − 27 breaks up the run of A bases present in the consensus TATA box, and the much weaker interaction with TAAAAAA, where the series of A bases is extended. In the latter case, the binding affinity and the degree of bending were both increased by the introduction of a second base-pair change to create the sequence TAAATAA, in which the A run was again interrupted. However, the relatively weak binding to that sequence, as well as the low affinity and decreased bend angle observed for TATTAAA, are harder to rationalize on the basis of this argument alone. The different energetics of DNA bending could still contribute to the observed differences in the preferred solution structures if, for example, deforming the DNA to the extent observed for the high-affinity complexes is more difficult for some sequences than for others due to steric constraints. In addition, TpA base steps have been proposed to be more ‘‘flexible’’ than other dinucleotide steps (reviewed by Travers & Klug, 1990), principally because TpA steps tend to unstack more readily. It is possible that the presence of this

JMB—MS 606 442 dinucleotide contributes to the stabilization of binding to some of the TATA boxes relative to others in which a TA was eliminated by a base-pair change. Although we observed a correlation between reduced bend angle and reduced affinity in this study, we do not necessarily expect that correlation will hold true for all TBP-DNA complexes. For example, we can imagine that the loss of a contact without perturbation of other features of the interaction (due, for example, to an amino acid change in the protein) could, depending on the magnitude of the effect, reduce the affinity without changing the preferred structure of the complex. In fact, such complexes, if they existed, could allow an estimate of the energy associated with the DNA deformation (i.e. how many kcal/mol of interaction free energy can be lost before the DNA can no longer be bent to the same extent). The correlation observed in this study may have come about, in part, because we used a set of mutations that were primarily selected to minimize changes to the minor groove surface with which the protein interacts (so, mostly A·T to T·A changes). Indeed, the two DNA sequence changes that introduced G·C base-pairs into the TATA box fit least well with the observed correlation. Those sequences, CATAAAA and TATAAAC, both formed complexes with longer lifetimes than was observed for other TATA boxes with similar apparent bend angles. However, in addition to containing G·C base-pairs, both of those sequences were altered at the positions of phenylalanine intercalation, where the specific base sequence might well influence the energy costs of kinking the DNA backbone. Such a mechanism of base sequence discrimination has been suggested for the SRY high mobility group (HMG) box, which has been proposed to bind within the minor groove and to partially insert an aliphatic side-chain between two adjacent base-pairs at the center of the DNA-binding site, which is bent in the complex (King & Weiss, 1993). The additional finding that CATAAAA and TATAAAC both promoted more transcription than expected based on the relative bend angles raises the interesting possibility that different structural features underlying an apparent reduction in bend angle (i.e. kink formation or tight contact with the minor groove surface) might lead to different functional consequences. In the above discussion, we have assumed that the gel mobility differences observed for the various complexes reflects an actual difference in the lowest free energy conformations. Another possible, although perhaps unlikely, interpretation is that the predominant forms of the complexes on consensus and non-consensus TATA boxes are nearly identical, with the apparent differences in bend angle reflecting the fraction of time spent in that complex. We cannot distinguish between these possibilities on the basis of the experiments presented here, because the gel methods we used to analyze the bend cannot distinguish between a homogeneous population of complexes containing DNA that is less bent than is optimal, and a heterogeneous population containing,

DNA Bending in TBP Recognition

in the extreme view, fully bent DNA in rapid equilibrium with unbent DNA. Instead, methods with which individual complexes can be visualized will be required. We have begun such a study using scanning force microscopy. Our initial results have indicated that the median bend angle observed for complexes bound to non-consensus TATA box sequences is significantly less than that observed for complexes bound at the consensus TATA box, although both types of complexes may be able to adopt a fairly wide range of conformations (S. Hermann, M. Guthold, C. Bustamante and D.K.H., unpublished observations). If the observed bend angles do reflect differences in the most stable structures of the various complexes, then additional structural information will be required to formulate a detailed picture of the complexes formed on non-consensus sites. In particular, it is not clear which features of the DNA conformation, the helix unwinding and/or the kinks due to phenylalanine intercalation, are altered in the complexes with non-consensus TATA boxes, as both would appear to be critical for TBP binding. The phasing analysis experiments (Figure 4) did not provide any clues, because they showed that the location and direction of the bend did not appear to change, even though the observed degree of bending varied among the different TATA box complexes. One possible solution to this apparent paradox is that the flexibility of the protein allows TBP to make similar protein-DNA contacts in all of the complexes even though the path of the DNA into and out of the complex is not identical. For example, the two DNA-binding domains of the protein may be able to alter their relative orientation to accommodate a reduced degree of helical unwinding while maintaining nearly optimal interactions with the minor groove surface of a non-consensus TATA sequence. This idea is supported by a comparison of the bound and unbound conformations of the Arabidopsis TBP, ˚ which showed that one domain was displaced 5 A relative to the other when the protein was bound to the consensus TATA box (J. L. Kim et al., 1993). In addition, Chasman et al. (1993) noted a small difference in the conformations of the two TBP molecules in the asymmetric unit of their yeast TBP crystal and suggested that this difference reflected a conformational flexibility of TBP that may be important to the interaction of the protein with the TATA box. An important contribution of DNA deformability to the binding energetics has been proposed for other proteins (reviewed by Steitz, 1990; Travers & Klug, 1990; Nussinov, 1990; Harrington, 1992). These include, most notably, nucleosomes (Drew & Travers, 1985; Satchwell et al., 1986), phage 434 repressor (Anderson et al., 1987; Koudelka et al., 1988), and the bacterial transcription activator CAP (Gartenberg & Crothers, 1988). For CAP, evidence of a correlation between the magnitude of bending and the affinity of a protein for its DNA-binding site was also observed. In that case, Gartenberg & Crothers (1988) showed that the protein-DNA complexes with the

JMB—MS 606 443

DNA Bending in TBP Recognition

lowest gel mobility, and presumably, therefore, the most highly bent conformation, contained sequences for which CAP had the highest affinity, similar to our observations with TBP complexes. However, the DNA sequences shown to influence both the affinity and the bending by CAP occurred in a region of DNA where CAP has been proposed to make only electrostatic contacts with DNA, flanking the DNA base-pairs that interact with CAP through hydrogen bonds (Schultz et al., 1991). In contrast, in this study of TBP binding, the base-pairs that were shown to influence the DNA conformation of the complex are the same as those that define the binding site. Our results showed that the bend angle correlates with the ability of TBP to promote basal transcription by pol II, suggesting that, directly or indirectly, the conformation of the complex is important in transcription initiation. Although the bending may be necessary only to optimize the TBP-DNA contacts, thereby increasing the occupancy of the TATA box, it is also possible that the TBP-induced DNA bend facilitates the binding of one or more additional basal transcription factors. The DNA bend may also promote the interaction of an activator with some other part of the general transcriptional machinery. To address the physiological significance of the observations reported here, it will be important to examine the binding and DNA-bending properties of TBP complexed with the other proteins with which it is associated in vivo. Initial experiments from our laboratory have shown that TFIID binding at the consensus TATA box appears to bend the DNA similarly to TBP (Lara Baxley and D.K.H., unpublished observations). TBP is also known to be involved in pol I and III transcription and has been found to be a component of TFIIIB, a pol III factor that has been shown to bend the DNA (Leveillard et al., 1991). Further studies like those presented here are

necessary in order to determine whether the DNA-bending properties of TBP are also important to pol I and III transcription.

Materials and Methods Protein purification Yeast TBP (yTBP) and the C-terminal domain of yTBP (yTBP(D3-60)) were purified as described (Hoopes et al., 1992). Recombinant human TBP (hTBP) was either purified by our own unpublished procedure or purchased from Promega.

Plasmid construction The pUCBend series of plasmids used in the circular permutation analysis was created as follows: pUCBend, kindly provided by Barbara Graves (University of Utah), contained the EcoRI-HindIII fragment of pBend (Kim et al., 1989) inserted into the EcoRI and HindIII sites of pUC13. pUCBend(WT) was constructed by restricting pWrm 18 (Hoopes et al., 1992) and isolating a DNA fragment containing the wild-type adenovirus major late promoter (Ad MLP) TATA box flanked by a XbaI site and a Klenow filled-in HindIII site. This fragment was ligated to XbaI-HincII-cut pUCBend. The other plasmids in the pUCBend series were created similarly, except that the DNA fragments used were obtained from the pWrm derivatives containing the appropriate TATA box mutations (our unpublished results). The plasmids containing TATA box mutations were given names that identify the base-pair change from the consensus sequence and the position of the mutation with respect to the start site of transcription from the ML promoter. Thus, a pUCBend plasmid containing CATAAAA (the 31C mutation) is called pUCBend(31C). The plasmids used in the phasing analysis were made in a series of steps. In the first step, the synthetic oligonucleotides shown below (Midland Oligos) were inserted into the XbaI site of pUCBend(WT).

JMB—MS 606 444 This step created a series of plasmids, designated pUCBend + 0(WT), pUCBend + 3(WT), etc., which contained the wild-type TATA box placed at varying distances from the center of three phased A-tracts (as shown in Figure 3A). In the second step, DNA fragments containing the MLP and the inserted oligonucleotides were isolated from the newly created pUCBend + (WT) plasmids following digestion with EcoRI and HindIII, and were ligated into pTZ18U. The purpose of this step was to permit subsequent preparation of single-stranded DNA for site-directed mutagenesis. Each of the resulting plasmids was then modified to facilitate the replacement of the wild-type TATA box with each of the mutant TATA boxes. This goal was accomplished in each case by creating an XbaI site at −61 (relative to the MLP start site) and, if necessary, destroying an existing XbaI site at the upstream side of the inserted oligonucleotide. This step created a series of plasmids, designated pB + 0WT, pB + 3WT, etc., each of which contains a unique XbaI site, located between the wild-type TATA box and the phased A-tracts. The mutant TATA boxes, isolated as 220 bp XbaI-HindIII fragments from the pUCBend(mutant) series, were then inserted into the XbaI-HindIII sites of these plasmids. The pB + 12WT plasmid was made by restricting pB + 8WT with XbaI, filling in using Klenow fragment, and religating. The pB + 2WT plasmid was created by ligating the +3 oligonucleotide into the XbaI site of pUCBend(WT) in the reverse orientation. DNA probes for gel mobility shift assays were labeled at the sites indicated in the Figure legends and were isolated as described (Starr & Hawley, 1991).

DNA-binding and gel mobility shift assays TBP binding conditions were as described (Starr & Hawley, 1991). For the binding reactions, 10 nM yTBP, hTBP or yTBP(D3-60) or buffer was incubated with the labeled DNA fragment in a total volume of 15 ml. All of the reactions contained 30 mg/ml poly[d(G-C)]. Final solution conditions included 12 mM Tris-HCl (pH 7.9 at 4°C), 20 mM Hepes (pH 8.4), 10 mM MgCl2 , 12% (v/v) glycerol, 60 mM KCl and 0.5 mM dithiothreitol. After 20 to 30 minutes at 30°C, reactions containing yTBP or yTBP(D3-60) were directly loaded onto a native 4% (w/v) polyacrylamide gel run in TGE buffer containing 5 mM MgCl2 (Hoopes et al., 1992). For hTBP, the reactions were incubated with 0.02% (w/v) Sarkosyl for three minutes at 30°C before loading to remove any non-specific hTBPTATA box complexes (Hoopes et al., 1992). Native gel electrophoresis was at 25 to 30 mA for six to eight hours at room temperature. We observed no difference between running the gels at 4°C and room temperature (data not shown). Subsequent processing of gels was as described (Starr & Hawley, 1991). When TATA box mutations were analyzed, the amount of labeled DNA fragment added was adjusted so that the total radioactivity of the DNA complexed with protein was approximately the same in all the reactions. The ratios when yTBP(D3-60) was used were as follows: WT, 1; 31C, 3; 30T, 1.5; 29A, 8; 29A27T, 5; 28T, 2; 27T28T, 1; 27T, 1; and 25C, 8.

Determination of the direction and magnitude of the angle of the TBP-induced DNA bend The direction of the DNA bend was determined by first approximating the location of the TBP-induced DNA bend by circular permutation analysis as described by

DNA Bending in TBP Recognition

Thompson & Landy (1988) and then using the phasing data to determine its direction (Zinkel & Crothers, 1987). The magnitude of the TBP-induced DNA bend was calculated using either the circular permutation or phasing analysis data. The circular permutation data of Figures 1 and 3 were analyzed essentially as described (Thompson & Landy, 1988) by applying the following formula: mM /mE = cos(ka/2) where mM is the mobility of the protein-DNA complex with the binding site in the middle of the DNA fragment, mE is the mobility of the protein-DNA complex with the binding site at the end of the DNA fragment, k is the coefficient to adjust for electrophoresis conditions, and a is the angle of the protein-induced DNA bend. A k-value of 0.51 was obtained using the intrinsic bend of 54° as a standard. This k-value is consistent with what others have obtained under similar gel conditions (Thompson & Landy, 1988). The magnitude of the TBP-induced DNA bend angles of the wild-type and all of the TATA box mutations were determined using this method. The magnitude of the TBP-induced DNA bend was also calculated from the circular permutation data by a second method that allows analysis of the electrophoretic mobilities of complexes in which the protein-binding site lies between the middle and the end of the DNA fragment (Ferrari et al., 1992). This method involves fitting the circular permutation data to the following quadratic equation: Rbound /Rfree = 2K(1 + cos u)(D/L)2 −2K(1 + cos u)(D/L) + K where u is the angle of the protein-induced DNA bend, K is a constant, D is the distance of the vertex of the angle u from the 5' end of the DNA fragment, and L is the length of the DNA fragment. A more complete derivation of this formula was given by Ferrari et al. (1992). This method was applied to TATAAAA, CATAAAA and TATTAAA. The angle of the TBP-induced DNA bend was independently determined using the phasing analysis described by Kerppola & Curran (1991a). The following equation was applied to the data: tan(kaB /2) =

mMax /mMin − 1 (mMax /mMin + 1)tan(kaC /2)

where aB is the angle of the protein-induced DNA bend, aC is the angle of the intrinsic DNA bend, k is the coefficient to adjust for electrophoretic conditions, and mMax and mMin are the maximum and minimum electrophoretic mobilities, observed for the DNA fragments in which the protein-induced bend most nearly counteracts (giving the maximum mobility) or most effectively cooperates with (giving the minimum mobility) the intrinsic bend. The intrinsic bend (see Figure 3A) was assumed to have an overall bend of 54° (18° per A-tract; Koo et al., 1990) and to be directed toward the minor groove at the centers of the A-tracts (Zinkel & Crothers, 1987; Salvo & Grindley, 1987).

Acknowledgements The authors thank Carlos Bustamante, Will McClure and Pete von Hippel for discussions and comments on the manuscript. This work was supported by grants from the National Institutes of Health (GM-46267) and the Searle Scholars Program of the Chicago Community Trust to D.K.H. D.B.S. was supported, in part, by a training grant from the National Institutes of Health.

JMB—MS 606 DNA Bending in TBP Recognition

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Edited by R. Schleif (Received 20 July 1994; accepted in revised form 11 April 1995)