TATA Box DNA Deformation with and without the TATA Box-binding Protein

Article No. jmbi.1999.2947 available online at http://www.idealibrary.com on

J. Mol. Biol. (1999) 291, 249±265

TATA Box DNA Deformation with and without the TATA Box-binding Protein Natalie A. Davis, Sangita S. Majee and Jason D. Kahn* Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742-2021, USA

DNA ring closure methods have been applied to TATA box DNA and its complex with the TATA box-binding protein (TBP). The J factors for cyclization (effective concentrations of one DNA end about the other) have been measured using cyclization kinetics, with and without bound TBP, for 18 DNA constructs containing the adenovirus major late promoter TATA box (TATAAAAG) separated by a variable helical phasing adapter from sequence-induced A-tract DNA bends. Six phasing lengths were used at three overall DNA lengths each. Cyclization kinetics were also measured in the absence of protein for the same set of molecules bearing a mutant TATA box (TACAAAAG). The results suggest that the TATA box DNA itself is strongly bent and anisotropically ¯exible, in a direction opposite to the bend induced by TBP, and that the mutant TACA box is much less bent/¯exible. The bending and ¯exibility of the free DNA may govern the energetics of recognition of different DNA sequences by TBP, and the intrinsic bend may act to repress transcription complex assembly in the absence of TBP. The cyclization kinetics of TBPDNA complexes in solution predict a geometry generally consistent with crystal structures, which show dramatic bending and unwinding. The novel observation of TBP-induced topoisomers suggests that this minicircle approach is able to distinguish TBP-induced unwinding from writhe (these cancel out in larger DNA), and this in turn suggests that changes in supercoiling in small topological domains can control TBP binding. # 1999 Academic Press

*Corresponding author

Keywords: DNA cyclization kinetics; DNA bending; DNA ¯exibility; DNA topology; writhe compensation

Introduction The TATA box-binding protein (TBP) is a sequence-speci®c DNA-binding protein required by all three eukaryotic RNA polymerases for transcription initiation. Its role in Pol II transcription is as part of the general transcription factor TFIID, Present address: N. Davis, Lombardi Cancer Center, Georgetown University Medical Center, Research Building, Suite W503, 3970 Reservoir Road, NW, Washington, DC 20007, USA. Abbreviations used: AdMLP, adenovirus major late promoter; CAP, catabolite activator protein; EGTA, ethylene glycol-bis(b-aminoethyl ether)-N,N,N0 ,N0 tetraacetic acid; J factor, Jacobson-Stockmayer factor; Lk, linking number; Tw, twist; Wr, writhe; TBP, TATA boxbinding protein; TAF, TBP-associated factor; TFIID, transcription factor II D; hTBP, human TBP; yTBP, yeast TBP; aTBP, Arabidopsis TBP. E-mail address of the corresponding author: [email protected] 0022-2836/99/320249±17 $30.00/0

which recognizes the TATA box in the promoter region. TBP alone can support basal transcription, but TBP-associated factors (TAFs) included in TFIID are required for activated transcription (Burley & Roeder, 1996; Hernandez, 1993). The crystal structures of several TBP
DNA complexes show that TBP binds to the minor groove of the TATA box, unwinds the TATA box DNA by approximately 105  , and bends the DNA towards the major groove by about 80  (Juo et al., 1996; Kim & Burley, 1994; Kim et al., 1993b; Nikolov et al., 1996). Much of the bending is due to Phe residues from the ``stirrups'' of the protein intercalating at the edges of the TATA box. The induced DNA structure is not changed substantially by the binding of general transcription factors TFIIB or TFIIA to the TBP
DNA complex (Geiger et al., 1996; Nikolov et al., 1995; Tan et al., 1996). TBP is an unusual DNA-binding protein, because proteins usually bind in the DNA major groove, and if the DNA bends it usually bends toward the protein (Pellegrini et al., 1995; Schultz et al., 1991). # 1999 Academic Press

250 TBP binds several variant TATA box sequences (Hahn et al., 1989; Wobbe & Struhl, 1990), and this observation as well as the paucity of direct hydrogen bonds to base-pairs led to the idea that recognition depends on DNA structure and deformability rather than sequence per se (Juo et al., 1996; Kim et al., 1993b). Hawley and co-workers have observed that the induced degree of bending of TATA box mutants correlates with the stability of DNA
TBP complexes (Starr et al., 1995), and variations in DNA
TBP complex structure may alter the function of TBP in transcription initiation (Diagana et al., 1997; Hoopes et al., 1998). Molecular mechanics and dynamics simulations suggest that the A ‡ T-rich TATA box DNA is readily deformed into the TBP-bound conformation, perhaps via an A-DNA like intermediate (Flatters et al., 1997; Lebrun et al., 1997; Pardo et al., 1998; Pastor et al., 1997), but the simulations did not show that the equilibrium TATA box structure bears any strong resemblance to the bound DNA shape. The TBP-induced bend is essential for TFIIB and TFIIA binding (Nikolov et al., 1995) and the assembly of the preinitiation complex (OelgeschlaÈger et al., 1996; Robert et al., 1998; Tang et al., 1996). Bending is often required for assembly of higherorder protein-DNA complexes (reviewed by Grosschedl, 1995). It may also promote transcription initiation by potentiating DNA looping (Santero et al., 1992), strengthening interactions between RNA polymerase and upstream DNA (Crothers & Steitz, 1992), storing energy (Ryu et al., 1994), or inducing allosteric changes in the DNA (Schurr et al., 1997). Monitoring changes in DNA bending and twisting during the transcription cycle would aid in studying all of these mechanisms. We set out to study assembly of TBP-containing complexes and changes in DNA structure during initiation using DNA cyclization, starting with the free DNA and the TBP
DNA complex. Cyclization allows the solution study of large-scale DNA properties, including ¯exibility, bending, and twist changes (Crothers et al., 1992; Kahn et al., 1994). In principle, it can be performed on complexes of arbitrary size, and the results can be simulated (De Santis et al., 1996; Kahn & Crothers, 1998; Manning et al., 1996). Here we report several unexpected results from this work. Cyclization ef®ciency is measured as the effective concentration of one aligned DNA end about the other, the J factor, where J  Kc/Ka is the ratio of the equilibrium constants for unimolecular cyclization, Kc, and bimolecular association, Ka (Flory et al., 1976; Jacobson & Stockmayer, 1950). J can also be measured by phage T4 DNA ligasemediated covalent ring closure kinetics: J ˆ k1/k2, where k1 is the cyclization rate constant and k2 is the bimolecular ligation rate constant (Kahn & Crothers, 1992; Koo et al., 1990; Shore & Baldwin, 1983a; Shore et al., 1981; Taylor & Hagerman, 1990). Protein induced bending decreases the DNA end-to-end distance and thus increases the ring clo-

TATA Box DNA Deformation with and without TBP

sure probability (Dripps & Wartell, 1987; Kotlarz et al., 1986), and multiple phased bends can additionally improve helix axis alignment at the ends to give much more dramatic effects (Hagerman & Ramadevi, 1990; Koo et al., 1990). Cyclization constructs containing two bends separated by a variable-length phasing adapter (Zinkel & Crothers, 1987) were developed to characterize catabolite activator protein (CAP)-induced bending, and in these molecules bending effects were ampli®ed to give about a 100,000-fold range in J factor (Kahn & Crothers, 1992). Monte Carlo simulations of the results give a solution geometry consistent with the co-crystal structure (Kahn & Crothers, 1998; Schultz et al., 1991). Finally, induced twisting and bending effects can be identi®ed independently by employing several isomers that differ in DNA torsion due to length changes outside the two sites of interest (Kahn et al., 1994). Parvin et al. (1995) used two TATA box-containing DNA constructs (derived from the original CAP site set) in a minicircle binding assay to show that TBP and TFIID bend DNA in the same direction as in co-crystal structures. TBP binds much more tightly to an in-phase than an out-of-phase minicircle, and the effect on binding af®nity is primarily due to very slow off-rates for in-phase circles, not to on-rates. They did not analyze twist effects. While minicircle binding is thermodynamically equivalent to cyclization kinetics as far as quantifying protein-induced distortion, it reports only on changes in DNA shape or ¯exibility, not on the properties of the free or the bound DNA independently. Grove et al. (1998) demonstrated similar enhanced thermodynamic stability but constant on-rates for TBP binding to TATA box DNA with deformable sites provided by loops or 5hydroxymethyluracil. The cyclization results presented here are the most extensive to date for a protein-DNA complex. They show that the unbound TATA box DNA is remarkably bent and/or anisotropically ¯exible and that this deformability is greatly reduced in a TATA box variant with decreased binding af®nity for TBP. The bend direction and magnitude for TBP are con®rmed to be similar to co-crystal structure results, but TBP unexpectedly introduces new topoisomers, probably due to protein-induced untwisting combined with minicircle cyclization constraints.

Results Bend phasing constructs for DNA cyclization We constructed six DNA molecules containing the adenovirus major late promoter (AdMLP) TATA box separated by a variable-length phasing adapter from a set of six phased A-tracts (Figure 1). This system was adapted from a set developed for a study of CAP-induced DNA bending (Kahn & Crothers, 1992, 1998) which has also been used to characterize conformational changes induced by

251

TATA Box DNA Deformation with and without TBP

Figure 1. Cyclization construct design and sequences. The sequence of 9T9 is shown in the context of pBluescript II KS (‡). The ClaI sites and capital letters demarcate the portion used for cyclization, which is ampli®ed by PCR with the indicated primers. The nomenclature is (®rst adapter #)(A/T)(second adapter #), where each number indicates the distance in base-pairs between the centers of the indicated EcoRI and MspI sites. The sequences inserted at the ®rst adapter are the reverse complements of those shown for the second adapter. The second adapter speci®es bend phasing in the linear starting material, and overall length variation is obtained by varying either the ®rst adapter or the downstream PCR primer; each row of Tables 1 and 2 represents a set in which the PCR primer was varied. The sequence between the brackets is inverted in xAy (versus xTy) constructs. The TATA box and TACA box are indicated by the box; only the eight base-pair TATAAAAG is identical with the AdMLP. The AvrII and MluI sites are unique in the plasmid. The second A-tract in from the second adapter in clone 17A11 has only ®ve A bases, which changes the length by one base-pair and alters bend phasing slightly. The total length ranges from 147-163 bp.

Fos/Jun (Sitlani & Crothers, 1996, 1998), GCN4 (Hockings et al., 1998), NF-kB (Kuprash et al., 1995), Myc/Max (McCormick et al., 1996), and TBP (Parvin et al., 1995). The TATA box sequence (TATAAAAG) was substituted for the CAP site using standard PCR and cloning methods. When a protein-induced bend is in-phase with the sequence-directed A-tract bend, the DNA will form a C shape and cyclize readily. If the two bends are out of phase, forming an S shape, cyclization will be slow. If there is no phase dependence, then the protein does not induce bending (there is controversy as to the lower limit of detection; see Hagerman, 1996; Kerppola, 1996; Sitlani & Crothers, 1996). These DNAs comprise about one persistence length (they range from 147163 bp), and therefore cyclization would be extremely slow were it not for the bends, and small structural changes (i.e. added bends) have marked effects on cyclization ef®ciency. A twist change shifts the optimum overall DNA length for cyclization: unwinding favors cyclization of longer lengths. Figure 2 shows initial computer models for the six constructs, based on the co-crystal structure of hTBP
DNA (Nikolov et al., 1996) and on wedge or junction models for sequence-directed DNA bending (Bolshoy et al., 1991; Koo et al., 1990). Figure 2(a) illustrates the phasing relationships, and Figure 2(b) and (c) show the expected bending in the free TATA box, which is directed into the minor groove at the center of the A-tract whereas TBP bends the DNA into the major groove. The two models for sequence-dependent DNA structure predict similar bend directions but different

magnitudes, and they predict much weaker bending for free than for bound DNA. Binding conditions and specificity The ideal conditions for DNA cyclization experiments are saturation of the speci®c site (here, the TATA box) with no non-speci®c binding. We characterized the af®nity and speci®city of TBP binding by electrophoretic mobility shift assay (Figure 3(a)), nitrocellulose ®lter binding, and DNase I footprinting (data not shown). The three assays showed that under our salt and competitor DNA conditions, apparent speci®c DNA binding saturates at about 30 % occupancy at 20-50 nM TBP; the lack of complete binding may be due to breakdown of the complex during digestion, loading, ®ltration, or electrophoresis (Hoopes et al., 1992). There is no evidence for non-speci®c binding in the gel shown in Figure 3(a). Speci®city was further con®rmed in mobility shift experiments with XbaI-restricted DNA (data not shown): only the TATA box fragment and not the A-tract fragment was observed to bind TBP. Also, only the TATA box was protected from DNase I at TBP concentrations below 100 nM. As an additional control, 20 nM TBP had little effect on the cyclization of TACA box variants, con®rming that interaction of TBP with the A-tracts or other sequences has no effect on the observed kinetics. Figure 3(b) shows time courses for DNA cyclization at varying TBP concentrations. The data show a transition from slow to fast cyclization as [TBP] increases, and the entire set was ®t with four parameters: cyclization rate constants for free and

252

TATA Box DNA Deformation with and without TBP

Figure 2. Modelling of DNA shapes. (a) Bend phasing constructs with bound TBP. DNA shapes were generated using the junction model for A-tracts (Koo et al., 1990) and averaged roll and tilt angles from the aTBP
DNA co-crystal structure (Kim & Burley, 1994), and otherwise assuming straight DNA. The six DNA constructs indicated were superimposed at the TATA box (see Figure 1 for nomenclature). The two views are identical superpositions rotated to illustrate the different bend phasings. The structure of hTBP
DNA (Nikolov et al., 1996) is superimposed on the TATA box. 9T9 and 15A9 differ slightly in phasing because A-tracts and T-tracts are not perfectly symmetrical. (b) For two free DNA constructs, the junction model for A-tracts (gray) is compared to the wedge angle model (colored) of Bolshoy et al. (1991), scaled to make the magnitude of the A-tract bend agree with experiment (Haran et al., 1994; Kahn & Crothers, 1998). The two models predict the same direction of bending around the TATA box but different bend magnitudes. (c) Scaled wedge angle model predictions for the six constructs, superimposed in the A-tract region. The predicted bend direction is opposite to that of the TBP-induced bend, but the bend magnitude is predicted to be much smaller than the protein-induced bend (20  versus 80  ). Figures were generated as described (Kahn & Crothers, 1998) and displayed in InsightII (MSI).

bound DNA, an active DNA concentration, and a dissociation constant (®t to be 34(6) nM) for TBP. The data analysis did not require consideration of

TBP reassortment during ligation, presumably because TBP dissociates and binds slowly. All the experiments suggest speci®c binding but not com-

253

TATA Box DNA Deformation with and without TBP

many species if the TBP concentration were high enough to give signi®cant non-speci®c binding. Single 30 minute time points at 10 nM and 50 nM TBP in most experiments showed that the amount of TBP-dependent products increases with TBP concentration, but that the results are qualitatively similar to the 20 nM kinetics. The low occupancy adds to the uncertainty in the J factors (see Materials and Methods). Cyclization kinetics results demonstrate DNA bending

Figure 3. TBP binding experiments: electrophoretic mobility shift and cyclization titration assays. (a) A mobility shift experiment on molecule 15A9 shows speci®c binding, with no smearing to lower mobility complexes until [TBP] exceeds 50 nM. Apparent binding saturates at 20-50 nM TBP under these conditions, with 30 % of the DNA bound but some dissociation visible in the gel. (b) The same samples were used in cyclization kinetics experiments at the indicated [TBP], 2 nM DNA, and 1000 units/ml ligase (4000 units/ml for free DNA, rescaled). All the data were ®t in MATLAB to a single model with four parameters: active DNA concentration, cyclization rate constants for free and bound DNA, and a TBP
DNA dissociation constant. 15A9 cyclizes rapidly so there is no bimolecular reaction. The data shows cyclization of mixtures increasingly composed of bound DNA, with a best-®t Kd of 34(6) nM, a cyclization rate constant 20-fold greater for bound than free, and 1 nM active DNA.

plete occupancy at 20-50 nM TBP. In particular, topoisomers (see below) were observed with DNA in excess over TBP (2 nM TBP, 4 nM DNA), so they are extremely unlikely to be multiple binding artifacts. Cyclization kinetics experiments typically were done at 20 nM TBP, at about 30 % occupancy of the TATA box. Keeping occupancy relatively low means that only two species (bound and free) contribute to the observed kinetics, as opposed to

J factors for 18 DNA molecules (three lengths for each of six phasings) were measured with and without TBP, essentially according to previously reported methods (Kahn & Crothers, 1992). The set covers 1.5 helical turns in length and a full turn in bend phasing. Typical autoradiograms of ligation time courses are shown in Figures 4 and 5, which illustrate that the cyclization kinetics results fall into two classes. Class I molecules cyclize slowly in the absence of TBP and rapidly in its presence, as expected from in-phase bending shown in Figure 2. Class II molecules cyclize relatively rapidly in the absence of TBP and generally more slowly in its presence, due to out-of-phase bending. This shows that the direction of TBP-induced bending agrees with crystal structures and previous minicircle studies (Parvin et al., 1995). We expected that the J factors here would be similar to those in the CAP work (Kahn & Crothers, 1992): the TATA box DNA differs at only ten positions from the CAP site constructs, and the overall 80  bend induced by TBP is similar to the 90  bend of CAP. This predicts free TATA box DNA J factors around 100 nM, with about a tenfold range of values for DNAs of equal length but different bend phasings; the MLP TATA box is an A-tract and thus should show up in this assay (Kahn & Crothers, 1998; Sitlani & Crothers, 1998). J factors and error estimates for the full set of constructs are given in Table 1. The most dif®cult problems in cyclization data analysis are in treating the effects of inactive DNA molecules and of incomplete occupancy (see Materials and Methods). All of the experiments reported here used DNA preparations which gave at least 50 % cyclization within 30 minutes, in the range where no more than a twofold error in the J factor is introduced (Hockings et al., 1998). The TBP occupancy could be estimated from the data when different topoisomers were formed. For other class I molecules, systematic errors due to low occupancy may lead to up to ca threefold underestimation of the J factors. The measurements are reproducible to within a factor of three or better. Our interpretations are not substantially affected by errors of this magnitude, as the J factors range from 1-28,000 nM and the qualitative appearance of topoisomers is the most critical aspect of the data. Rapid cyclization of free DNA in some class II ‡ TBP cases made it impossible to measure accu-

254

TATA Box DNA Deformation with and without TBP

Figure 4. Cyclization kinetics of a class I construct: strong enhancement of slow cyclization and appearance of a negative topoisomer. (a) The autoradiogram shows cyclization kinetics for the 20A11 construct with and without 20 nM TBP. In the presence of TBP a new negative topoisomer is formed which builds up rapidly, but plateaus after 15 minutes due to incomplete TBP occupancy. Time points are at 0, 1, 2, 3, 5, 15, and 30 minutes, with 30 minute time points for 10 and 50 nM TBP on the sides. [DNA] was 2 mM, ligase concentration was 1000 units/ml with TBP, 4000 units/ml without TBP (the observed activity ratio was close to 1:1 in this particular reaction). The weak band above the negative supercoil is a mobility-shifted TBP
circle complex that survives protease K treatment but is sensitive to phenol. (b) The kinetics shown in (a) were analyzed using MATLAB to treat all the data simultaneously. The ®t parameters are two cyclization rate constants (bound and free), relative ligase activity, TBP occupancy (here, 20 %), and a single bimolecular ligation rate constant, for a total of ®ve parameters for ®ve curves. The calculated J factors are 110 nM for the TBP-induced
Lk ˆ ÿ 1 topoisomer and 8.1 nM for the free DNA
Lk ˆ 0 topoisomer.

rate values for the bound DNA: the numerical values in Table 1 are only upper bounds and were not used further. TATA box DNA is strongly bent and/or anisotropically flexible Some of the J factors in Table 1 are strikingly different from our expectations. The free TATA box DNA exhibits J factors ranging from 1.4 to 6600 nM, corresponding to bending of about 60  (Kahn & Crothers, 1998), versus 18  for an A-tract. The bend phase dependence of the free DNA J factors demonstrated that the bend direction was generally opposite to that induced by TBP, in agreement with the models in Figure 2: molecules which cyclize ef®ciently in the presence of TBP cyclize slowly in its absence and vice versa. We con®rmed that the unexpected cyclization results were due to the TATA box and not to adventitious bending elsewhere by substituting

TACAAAAG for TATAAAAG in each of the constructs. Ligation kinetics on the 18 ``TACA box'' length and phasing variants (Table 2) shows that the free TACA box behaves as expected for a simple A-tract. It supports less ef®cient cyclization and gives only a 20-fold modulation in the J factor due to bending (taking ratios of the maximum J factors for each bend phasing), versus at least 100fold for the TATA box. The TACA box did not support speci®c enough TBP binding to allow J factor measurements, but spot checks of the TACA kinetics at >50 nM TBP were qualitatively similar to TBP
TATA at 20 nM TBP (data not shown). TACAAAG supports a signi®cant level of transcription (20 %) relative to TATAAAG, but activity is higher for HeLa TFIID than for yTBP (Wobbe & Struhl, 1990), suggesting that TAFs might enhance binding to this sequence. The effect of the TATA sequence could be due to static bending, anisotropic ¯exibility, or both. Surprisingly, preliminary ligation ladder experiments

TATA Box DNA Deformation with and without TBP

255

Figure 5. Cyclization kinetics of class II constructs: weak repression of rapid cyclization. (a) The autoradiogram shows cyclization of 8T15 in the presence and absence of TBP. Both sets of reactions were done at 2 nM DNA and 20 units/ml ligase (50-fold less than in Figure 4), and with and without TBP the J factors are very high (6000 nM). No other products were observed, as cyclization is much more rapid than bimolecular ligation. (b) Quanti®cation shows that TBP reduces the cyclization rate slightly. This is ®t partly as a 12 % decrease in active DNA concentration and partly as a decreased cyclization rate constant, and thus an accurate value for the ‡TBP J factor cannot be obtained.

(Harrington, 1993; Ulanovsky et al., 1986) show that the TATA box behaves like a simple A-tract (K. Kang and J.D.K. unpublished results), suggesting added ¯exibility instead of a large static bend. Further evidence for the lack of a large static bend comes from TATA/TACA comparisons: a static bend should repress TATA cyclization when outof-phase with the A-tracts, and the J factors should be less than the corresponding TACA construct; we observe instead that the TATA J factors are almost always larger than those for TACA. The putative bending/¯exibility must be anisotropic because otherwise the observed strong cyclization phase dependence would be averaged out over all bend orientations. Anisotropy is supported by subtle phase-dependent changes in the relative J factors between TATA and TACA fragments, with the class I J factors being on average ®vefold larger and the class II J factors tenfold larger for TATA/ TACA (geometric means). Flexibility rather than static bending is also suggested by the weak dependence of cyclization on bend phasing: class II molecules 10A13, 8T15, and 8A17 all have similar J factors even though the bend phasing varies over

about 120  (see Table 1 and Figure 2). The TACA 11T15C showed slightly better cyclization than 13A13C or 11A17C, suggesting decreased ¯exibility. The class II molecules have a sequence-directed TATA box bend in-phase with the A-tract bend. Their J factors are strongly dependent on overall DNA length at constant bend phasing (e.g. J decreases 5000-fold for 10A13-13A13-16A13 as the overall length moves away from an integral number of helical turns). In contrast, the class I molecules, with out-of-phase bends, show much weaker length dependence, only ®ve- to 40-fold. Cyclization of molecules with large bends requires bend alignment via twist changes that bring the bends into phase, rather than changes in local bend direction (Kahn & Crothers, 1998). If this holds for the TATA box bend, then the length-dependence results could be due to bend alignment combined with coupling between bending (or anisotropic bending ¯exibility) and torsional ¯exibility. Thus, for class II molecules the bends are in-phase, and torsional changes in the intervening DNA would move the bends out-of-phase and prevent cycliza-

256

TATA Box DNA Deformation with and without TBP

Table 1. J factors for cyclization of TATA box-A-tract constructs with and without TBP A. Class I: in-phase A-tract and TBP bends Phasing

Turnsd

147 bp 14.14

47 bpc

4.50

2.4  0.6b

2.4  1.6

(9)T9 ‡ TBP (15)A9 (15)A9 ‡ TBP

47 47 47

4.50 4.50 4.50

7700  2100

440  130

(17)A11 (17)A11 ‡ TBP

49 49

4.69 4.69

Construct (9)aT9

Total L

150 14.42

153 14.71

13  3 (0) 1.7  0.7 (ÿ)e 140  50 (ÿ)f 1.4 6.9 (0) 5.3 (ÿ)

B. Class II: out-of-phase A-tract and TBP bends Phasing

Turnsd

(13)A13

51

4.88

6600  900

1200  700

(13)A13 ‡ TBP

51

4.88

(3200  1000)g

(1200  500)g

(11)T15 (11)T15 ‡ TBP

53 53

5.07 5.07

5800  200 (4600  400)g

3500  700 (2200  60)g

(11)A17

55

5.26

(11)A17 ‡ TBP

55

158 15.19

6500  1500 g

5.26

(4000  400)

159 15.29

34  3 710  150

64  12 28,000  3000

54  3 130  30

14  5 1200  6

7.8  0.3 150  40 (ÿ)f

161 15.48

163 15.67

Total L

155 14.90

Construct

157 15.09

156 15.00

160 15.38

3700  1200

162 15.57

1.1  0.2 (0) 1.3  0.3 (ÿ) 1.0  0.4 (0) 4.3  0.7 (ÿ) 8.5  0.8 67  40 (0) 13  4 (‡)

g

(2900  400)

1.6  0.1 (0) 1.2  0.1 (ÿ) 840  300 (0)f 270  80 (ÿ)f

a

(n) indicates total length was varied via PCR (outside the bends) to give n  3 as well as the parent molecule. All J factors are in nM. The mean of at least two determinations  the standard error of the mean is given. J factors for 12A9 were determined quantitatively only once, but the ÿ1 topoisomer was reproducibly observed in qualitative experiments. c Distance from center of TATA box to center of ®rst A-tract: half-integral turns indicates in-phase TBP-A-tract bending. d Calculations assume B-DNA helical repeat ˆ 10.45 bp/turn, A-tracts 10.33 bp/turn, no allowance for TBP unwinding. e Where (0), (ÿ), or (‡) are indicated, they accompany the J factors of the corresponding topoisomers. The (0) topoisomer is the predominant topoisomer observed in the absence of TBP. The positive topoisomer for 11T15 was not always observed. f These values were ®t with explicit consideration of site occupancy, and therefore are not lower bounds. Other ‡TBP values do not consider occupancy. For class I, the true values could be as much as threefold higher. g Values in parentheses are upper bounds. Actual values may be signi®cantly lower due to low occupancy of TATA by TBP. b

Table 2. J factors for cyclization of variant TACA box-A-tract constructs without TBP A. Class I: TACA box - A-tract constructs Construct

Phasing

Turnsd

(9)aT9C (15)A9C (17)A11C

47 bpc 47 49

4.50 4.50 4.69

147 bp 14.14

150 14.42

153 14.71

0.76  0.07b

1.1  0.3

0.71  0.14 1.8  0.3

B. Class II: TACA box - A-tract constructs d

155 14.90

Construct

Phasing

Turns

(13)A13C

51

4.88

410  140

(11)T15C (11)A17C

53 55

5.07 5.26

640  140

157 15.09

158 15.19

Total L 156 15.00 10  0.4 16  5 Total L 160 15.38

54  19 320  7

250  20

29  2

159 15.29

162 15.57

2.8  0.3 3.2  0.1

0.42  0.01

161 15.48

163 15.67

0.68  0.07 (0)e 0.21  0.01 (ÿ) 1.9  0.6

0.38  0.11 (0) 0.49  0.12 (ÿ)

a (n) indicates total length was varied via PCR (outside the bends) to give n  3 as well as the parent molecule. The C indicates TACA box. b All J factors are in nM and are the mean  the standard error of the mean for two independent determinations. c Distance from center of TACA box to center of ®rst A-tract. d Calculations assume B-DNA helical repeat ˆ 10.45 bp/turn, A-tracts 10.33 bp/turn, no allowance for TBP unwinding. e Where (0) or (ÿ) are indicated, they accompany the J factors of the corresponding topoisomers. The linking number for the (ÿ) topoisomer is presumably the same as that of the shorter members of the set.

257

TATA Box DNA Deformation with and without TBP

tion. Strong length dependence arises because the DNA segment which can twist to bring the DNA ends into torsional alignment is very short, being the only DNA outside the two bends, and a large twist change over a short distance has a large free energy cost. Bend alignment alone does not predict the observed weak length (torsion) dependence for class I molecules, for which we propose two mechanisms. First, if the TATA box bend is removed or inverted, the entire DNA can participate in torsional changes needed to bring the ends into torsional alignment. However, whereas the predicted length dependence is still 50-fold for 156 bp minicircles based on previous experiments with similar systems (Hockings et al., 1998; Kahn et al., 1994; Sitlani & Crothers, 1996), we observe only ®ve- to 40-fold modulation. Weak torsional modulation in class I may be due to torsional swiveling in the TATA box when it is deformed away from its equilibrium bend. Theories for twist-bend coupling suggest that DNA bend deformation will increase torsional ¯exibility (Marko & Siggia, 1994). In this view, the decrease in the overall J factors for class I versus class II is due to the free energy cost of bend deformation, but once this cost is paid the torsion is free. In summary, we propose that anisotropic ¯exibility of the TATA box as well as some bending explains strongly phase-dependent cyclization enhancement for these molecules. Length dependence trends in the data suggest that deforming the TATA box bend increases torsional ¯exibility. The TACA box has qualitatively similar but much weaker effects on DNA cyclization.

TBP-dependent topological changes upon cyclization The cyclization probability for class I DNA (TBP and A-tract bends in-phase) increased upon TBP binding, but there was an unexpected accompanying increase in the optimum DNA length for cyclization, consistent with DNA unwinding. Surprisingly, TBP binding to several different molecules in both classes also induced the formation of new topoisomers upon cyclization, which was not anticipated because TBP alone generally does not cause topological changes in plasmid relaxation assays (Lorch & Kornberg, 1993; OelgeschlaÈger et al., 1996). Our constructs are much too small to give a distribution of topoisomers; only one or at most two topoisomers are energetically accessible (Shore & Baldwin, 1983b). Topoisomers were identi®ed by comparison with negative topoisomers obtained by ligation in the presence of ethidium (Figure 6 and Table 1). When the TBP and A-tract bends are in phase (class I), only
Lk ˆ ÿ 1 topoisomers form. For out-of-phase molecules the results are more complicated: a
Lk ˆ ÿ 1 topoisomer was observed for 16A13, enhancement of cyclization of both the
Lk ˆ 0 and the
Lk ˆ ÿ 1 topoisomers for 14A17, and a
Lk ˆ ‡ 1 topoisomer for 14T15 (based on increased mobility in an ethidium gel and failure to comigrate with standards). We veri®ed the authenticity of all the topoisomers by Bal31 digestion and by isolation and re-analysis of the DNA, which showed the same mobilities (data not shown). These topological results and the contrast with plasmid experiments can all be rationalized by con-

Figure 6. Identi®cation of minicircle topoisomers. The autoradiogram shows 30 minute reactions performed at two TBP concentrations compared to negative supercoil standards generated by cyclization in the presence of ethidium. The gel contained 0.1 mg/ml ethidium, so negative topoisomers are relaxed and run more slowly, positive topoisomers run more rapidly as indicated. The second and sixth lanes of each set are duplicate reactions. Molecule 14T15 gives a new positive topoisomer but the cyclization of the original molecule is also enhanced, 14A17 shows primarily enhancement of the original topoisomer with a weaker effect on the negative topoisomer, and 16A13 shows weak enhancement primarily of the negative topoisomer. [DNA] was 2 nM.

258

TATA Box DNA Deformation with and without TBP

Figure 7. Writhe compensation in minicircles versus plasmids. (a) The local writhe induced by TBP binding. On the left, the co-crystal structure of human TBP bound to DNA (Nikolov et al., 1996) shows the DNA helix axis projecting out of the page on the left side of the complex and into the page on the right side. The pair of diagrams on the right show the co-complex structure superimposed on schematic models for plasmid DNA (left, red) and minicircle DNA (right, magenta). The crossover represents the local positive superhelical writhe contribution induced by TBP. (b) Minicircle-speci®c writhe compensation. In large, ¯exible plasmid DNA (left, red) the TBP-induced bend induces a neighboring crossover of either sign, and TBP's local writhe contribution adds to the overall writhe of the plasmid. The shape of the plasmid DNA is arbitrary. In a minicircle (right, magenta), in contrast, the diagram illustrates that the protein induces much less additional bending relative to free minicircle and that cyclization requires compensating negative writhe (sketched at the lower right) to maintain a nearly ¯at shape for the circle. These effects reduce the change in writhe due to TBP. The Figure was created in InsightII (MSI), with DNA pseudoatom positions from equations for the space curves.

sidering the TBP
DNA structure and the process of minicircle formation. Figure 7 illustrates proposed consequences of the minicircle's small size and minimal writhe (Frank-Kamenetskii, 1990). In plasmids, TBP-induced unwinding (negative
Tw) and writhe (bending leading to a positive
Wr contribution) combine to give a linking number change
Lk 
Tw ‡
Wr  0 (Kim et al., 1993a,b). The TBP-induced bend should localize to the end of an apical DNA loop (Laundon & Grif®th, 1988) and the positive writhe contribution is then realized within the loop. In contrast, TBP in a minicircle adds much less to total bending and thus gives less change in writhe, while the

unbound DNA also adds a small negative writhe contribution. Numerical computation of writhe for simple models also suggests that relatively subtle ¯attening of the TBP-bound TATA box could give a signi®cantly decreased writhe (data not shown). Regardless of the detailed mechanism of writhe cancellation, the experimental results suggest that the free and bound circles have nearly the same, very small, writhe. TBP-induced negative
Tw is then not cancelled out, and the result is a net negative
Lk 
Tw in the bound minicircle relative to the minicircle formed without TBP. The induced
Lk can be estimated from the change in the optimum cyclization length and

259

TATA Box DNA Deformation with and without TBP

from the lengths giving new class I topoisomers. The 15A9 and 17A11 sets cyclize optimally at about 15.3 helical turns, for
Lk  ÿ 0.3 (this value ®ts the data as a whole the best), and the 9T9 set gives about 14.14 turns. (These values depend on DNA helical repeats from previous work, but our optimum cyclization lengths without TBP are correctly predicted at 15.0(0.1) helical turns.) The
Lk changes the observed integral linking number only when the free linear DNA Lk0 (ˆTw0 ˆ length/helical repeat) is such that Lkm (the integer nearest Lk0) changes upon TBP binding (for de®nitions, see Bates & Maxwell, 1993). For example, free linear 12A9 is about Lk0 ˆ 14.7 turns of DNA, and the Lk ˆ Lkm of the free 12A9 minicircle is 15. With TBP bound, 12A9 is unwound to about 14.7 ÿ 0.3 ˆ 14.4 turns and cyclizes to give integral Lk ˆ 14, a new negative topoisomer. In contrast, for example, 17A11 is 15.3 turns and cyclizes at Lk ˆ 15 with or without the negative
Lk from TBP. This predicts that molecules that switch topology with TBP are torsionally non-optimal both with and without protein. This is clear in the data: the largest J factor for a topoisomer is about 270 nM for bound 14A17 versus 28,000 nM for bound in-phase 18A9. For class II molecules, bend alignment imposes a twist constraint, as discussed above. For 14A17 and 14T15, the required overwinding of the interbend DNA cancels TBP-induced negative
Tw (see Figure 2). For 14A17, this leads to no net linking number change, and TBP enhances cyclization of both of the original topoisomers. For 14T15, the larger positive
Tw required for bend alignment can overbalance TBP unwinding and give a new positive topoisomer. For 16A13, bend alignment unwinding and TBP induced unwinding should be additive. Weak enhancement of cyclization is observed, perhaps because the molecule has been unwound so much that it is out of phase at 14.8 turns. Comparison with co-crystal structures of TBP
DNA The J factors for TBP
DNA complexes fall in the same range (1-28,000 nM) as those measured for CAP
DNA complexes (1-20,000 nM), con®rming that the two proteins' long-range effects on DNA structure are similar, though their detailed structures are quite different. However, the TBP J factors may be underestimated up to threefold due to substoichiometric protein occupancy in our experiments, and the out-of-phase molecules appear to cyclize with higher ef®ciency than the corresponding CAP molecules. Increased cyclization ef®ciency would suggest a modestly larger bend angle (110  , based on Figure 5 by Kahn & Crothers, 1998) or increased ¯exibility in the TBP
DNA complex. Variations in the position or 50 ! 30 orientation of TBP (Cox et al., 1997) would contribute to weaker phasing amplitude rather than giving an absolute increase in J factor. Twisting the b-sheet

recognition surface of TBP could increase the apparent bend angle by making the overall bend more planar (this would also contribute to writhe cancellation as above), and this deformation occurs in molecular dynamics simulations of the complex (Miaskiewicz & Ornstein, 1996, their Figure 3). Monte Carlo and rod mechanics methods (Kahn & Crothers, 1998; Manning et al., 1996) are being applied to these data (data not shown); the correct prediction of topological changes will be a stringent test for the simulation methods.

Discussion Cyclization kinetics measurements on bend phasing constructs containing A-tracts and TATA boxes, with and without TBP, show that the solution structure of the TBP
DNA complex is similar in bend direction and magnitude to the co-crystal. They have also yielded two surprising results relevant to the mechanism of TBP binding. First, remarkable bending and/or ¯exibility of the AdMLP TATA box DNA suggest that the conformation and deformability of the free DNA is important in the recognition of the TATA box. Second, the unexpected production of minicircle topoisomers in cyclization of TBP
DNA suggests that TBP-induced twist changes, previously thought to be generally compensated by writhe, may be relevant to the mechanism of TBP binding and recognition. Role of the TATA box in the mechanism of TBP recognition The bending or ¯exibility of the TATA box DNA was demonstrated by its effect on cyclization kinetics. Free DNA with the TATAAAAG sequence cyclized with an ef®ciency to be expected for a 60  bend (Kahn & Crothers, 1998), and phasing analysis suggested that the bend is directed toward the minor groove at the center of the TATA box, in the opposite direction to the TBP-induced bend but in the direction expected for an A-tract. There also appears to be a range of accessible bend directions, and combined with the absence of an effect in ligation ladder experiments this suggests that the main component of the enhanced ¯exibility is directed perpendicular to the static A-tract bend. The set of TACA box variants, in which one of the 160 bp in each of the minicircle constructs was changed, showed cyclization properties more similar to previous results on CAP site DNA or single A-tracts, suggesting that the TACAAAAG sequence behaves more like a normal A-tract. The TACA box bending or ¯exibility is in the same direction as for the TATA box but much weaker in magnitude. Our results do not provide a molecular-level explanation for the TATA-induced ¯exibility, and the results are dif®cult to reconcile with nearestneighbor models for DNA conformation, which would not predict such a strong effect from a

260 single base change. Non-local bending has previously been suggested for AP-1 sites versus CREB sites (Hockings et al., 1998; Paolella et al., 1994). However, we do note that bending by roll on each side of the second T residue (which when changed to a C residue abolishes the effect) would give the observed direction of anisotropic ¯exibility, roughly perpendicular to the bend induced by the neighboring A-tract. Dramatic compression of the major groove at this position has been observed in molecular dynamics simulations (Flatters et al., 1997), and increased roll ¯exibility at YR steps has been identi®ed as a determinant for proteininduced bending (Suzuki & Yagi, 1995). The TATA tetranucleotide is over-represented in DNA sequences selected for high-af®nity nucleosome binding (Widlund et al., 1997). Coupled with only weak enhancement of the A-tract bend, this ¯exible roll model may explain increased cyclization ef®ciency for all three class II molecules as well as the ligation ladder results. It is possible that the TATA box exists in two conformational states separated by an energy barrier, one of which is highly bent/¯exible as above but rarely populated. The ligation assay is sensitive to this small transient population if the ¯exed state has a large J factor (due to phasing against the large static A-tract bend), but a rare ¯exed state might not enhance cyclization of multimers since most of the TATA boxes would not be ¯exed. The TATA box also might not appear unusual to bulk probes such as ¯uorescence (Parkhurst et al., 1996; Perez-Howard et al., 1995). The sequence TATAAT displays elevated hydrogen exchange rates and pre-melting conformational changes in NMR experiments, indicative of transient deformations (Patel et al., 1985). Several groups have proposed that the TATA box may be predisposed to adopting the TBPbound conformation. Our results agree with the idea that DNA conformational predilections are important to recognition, but differ sharply in the speci®cs of how this idea might be realized. Sigler and co-workers suggested that the ¯exibility of the TATA part of the TATA box might contribute to recognition and polarity (Kim et al., 1993b). Shakked and co-workers have proposed that rotating the bases about the glycosidic bond or pulling on the edges of the TATA box will deform the DNA into a structure similar to the bound shape (Guzikevich-Guerstein & Shakked, 1996; Lebrun et al., 1997), and molecular dynamics suggests that the bound conformation is accessible from A-DNA (Pardo et al., 1998). Based on these ideas, our experiments might have been expected to demonstrate pre-bending in the same direction as the TBP-induced bend, as has been observed for CAP. In fact, the bend appears to be in the opposite direction, in the direction expected for an A-tract. We suggest that the ¯exed state reorients upon protein binding, and that in this way the DNA is energetically/kinetically predisposed to adopt the TBP-bound conformation but that structurally the

TATA Box DNA Deformation with and without TBP

free DNA does not resemble the bound form. This could suppress aberrant assembly of transcription complexes in the absence of TBP, as potential interacting partners would be separated rather than brought together: the TATA box-TBP system would have the character of a binary switch for transcription. Lebrun et al. (1997) showed that DNA stretching could induce a conformation like the bound shape, but they noted that this did not require ground-state deformation of the DNA; we are extending their idea that a free energy valley connects the two conformations to the idea that the low point of the valley lies on the other side of canonical DNA. The TACAAAAG sequence does not have the same propensity and is bound much more poorly, even though the bound structure is very similar to TATAAAAG
TBP (S. Burley, personal communication). We propose that the function of the free DNA bend is to provide a lever arm for TBP to unwind the DNA: after the protein binds the initial bent state, reorientation of the bend helps unwind the DNA. TBP has been observed to bind DNA about 100-fold more slowly than the diffusion controlled rate (Hoopes et al., 1992; Petri et al., 1995). The molecular origin of slow binding has been proposed to be: (1) slow conversion of a TBP
DNA encounter complex to a ®nal stable complex (Hoopes et al., 1998); (2) rapid TBP binding to a rare population of transiently pre-bent DNA (Parkhurst et al., 1996); (3) slow dissociation of dimeric TBP to form monomers that bind DNA rapidly (Coleman et al., 1995); or (4) non-speci®c TBP
DNA binding followed by slow sliding to ®nd a speci®c site (Coleman & Pugh, 1995). Our proposal combines elements of mechanisms (1) and (2). Rate-limiting bend reorientation explains why pre-bending and pre-¯exing affect off-rates much more than on-rates (Grove et al., 1998; Parvin et al., 1995). Pre-bending the TATA box toward the major groove is opposite to its preferred direction, and if productive binding requires re-establishing a lever arm it would still be slow. This brings up the general point that from the extent of pre-bending in a circle one cannot deduce the overall effect of pre-bending on binding. The crucial variable is the difference in deformation free energy required to form the transition state or the ®nal bound shape in linear and circular DNA (Kahn & Crothers, 1993). Implications of TBP-induced topological changes The observation of TBP-induced topoisomers shows that topological effects that are undetectable in plasmid experiments can appear when minicircles are used. Topological relaxation experiments with TATA box plasmids have generally shown that TBP alone does not induce topological change in spite of the dramatic induced DNA unwinding. (Reconstituted TFIID induces topological unwinding believed to be due to DNA wrapping (Lorch &

261

TATA Box DNA Deformation with and without TBP

Kornberg, 1993; OelgeschlaÈger et al., 1996), and Tabuchi et al. (1993) observed a very low level of negative supercoiling with TBP.) The lack of topological change seemed to disallow the possibility of coupling topological change and the binding of TBP alone. We assert that while this twist-writhe compensation is feasible in the context of plasmidsized DNA in a minicircle or other constrained DNA the cyclization process requires a near-zero writhe, canceling TBP-induced writhe and revealing TBP's unwinding effect (see Results). If TBP binding induces negative supercoiling, then TBP should bind with a higher degree of af®nity to negatively supercoiled DNA because the relaxation of such supercoiling (writhe) will provide a thermodynamic driving force. (Preliminary gel mobility shift experiments have shown that this is the case; data not shown.) Enhanced binding to negative supercoils might also predict enhanced transcription of negatively supercoiled templates, especially if TFIID binding is rate limiting (Mizutani et al., 1991), but supercoiling could affect many other steps in complicated ways. Repressed transcription on a minicircle template (TenHarmsel & Biggin, 1995) could be due to increased bending strain upon DNA wrapping around TFIID or later intermediates in transcription complex assembly (Tang et al., 1996). In summary, our results on the intrinsic shape and deformability of the TATA box show that DNA deformability is important in recognition, but that the ground-state of the DNA need not resemble the target state. The minicircle approach can detect topological effects hidden from standard methods, and minicircles may provide model systems for small topological domains in larger DNA.

Materials and Methods Proteins, enzymes and materials Yeast TBP (yTBP) and Arabidopsis TBP2 (aTBP), generously supplied by Dr Michael Brenowitz and Dr Stephen Burley, respectively, were stored in 20 mM Hepes (pH 8), 50 mM KCl, 10 mM DTT, 0.1 mM EDTA and 50 % (v/v) glycerol at ÿ20  C. Spot-checks showed identical cyclization results for the two proteins; the experiments reported here were done using yTBP. Phage T4 DNA ligase and all restriction enzymes were from New England Biolabs (NEB), AmpliTaq was from PerkinElmer, radionucleotides from NEN, and DNA oligonucleotides from Keystone or Gibco/BRL. Buffer A, used for all experiments containing TBP, is 50 mM Hepes (pH 8), 90 mM potassium glutamate, 5 mM EGTA, 2.5 mM DTT, 10 % (v/v) glycerol, 10 mM Mg(OAc)2, 100 mg/ml gelatin, 2.5 mg/ml poly(dI-dC), and 0.5 % (v/v) Nonidet P-40 (NP-40). DNA constructs The phased TATA box-A-tract DNAs were derived from CAP site-A-tract constructs (Kahn & Crothers, 1992, 1998). The CAP site was replaced with the AdMLP TATA box (without the native ¯anking sequences) using standard PCR methods. The resulting DNA fragments

were cloned as XhoI-HindIII fragments into a modi®ed pBluescript II KS (‡) vector (Stratagene) to give the set shown in Figure 1. All clones were veri®ed by dideoxy sequencing. Advantages of the new set are that: (1) inserts are directionally cloned; (2) the TATA box can be excised as a 36 bp MluI-AvrII fragment without affecting other sequences; (3) inserts can be excised by ClaI for direct use as cyclization substrates; and (4) PCR primers used for substrate production do not overlap the TATA box. The TACA box inserts were produced by mutually primed Klenow extension of oligonucleotides, followed by restriction with MluI and StyI and cloning into MluI/ AvrII-cut parent plasmids. The TACA box DNAs differ from the parent molecules at a single base-pair. Synthesis of cyclization substrates BstNI-restricted plasmids were used as PCR templates (106 molecules/ml) in 150 ml reactions containing 0.2 mM each primer, 200 mM each dNTP, 6 units of Amplitaq, 10 % (v/v) glycerol, 20 mCi of [a-32P]dATP, 50 mM KCl, 10 mM Tris-HCl (pH 8.4) and 2 mM MgCl2. The mixtures were subjected to 25 cycles of 30 seconds at 94  C, 30 seconds at 55  C, one second at 60  C, one second at 66  C, one minute at 72  C, followed by a ®nal extension step of ®ve minutes at 72  C. The upstream primer is 50 -GGAAACAGCTATGACCATGATTACGCC (ERP, an extended M13 reverse primer). The downstream primer is 50 -GCAGTAAGCTTATCGATTCCATGGCGCAACGCGTTTAA (DHIPT), the ÿ3 primer 50 -AAGCTTATCGATTCCATGGCACGCGTTTAA, or the ‡3 primer 50 -AAGCTTATCGATTCCATGGCATAGCAACGCGTTTAA. PCR products were phenol/chloroform-extracted, precipitated, resuspended, and incubated overnight at 37  C with 15 units of ClaI in 80 ml. Cyclization substrates were puri®ed on 8 % native gels (40:1 acrylamide/bis, 50 mM Tris borate, 1 mM EDTA), eluted, and precipitated. DNA concentrations were estimated by scintillation counting, using the speci®c activity of the dATP. Electrophoretic mobility shift assays DNA (4 nM) was incubated for 30 minutes at 30  C with 0-100 nM yTBP in buffer A. Protein/DNA complexes were analyzed on an 8 % native gel (75:1 acrylamide/bis, 50 mM Tris borate, 1 mM EDTA, 2 mM DTT, 3 mM MgCl2, 1.1 % glycerol, thermostatted at 30  C in a Hoefer SE 600 apparatus, run at 24 V/cm in TBE running buffer containing 3 mM MgCl2). Dried gels were analyzed using a Molecular Dynamics PhosphorImager. Cyclization kinetics measurements Time courses were performed at 0-50 nM yTBP and 2-8 nM DNA. yTBP was allowed to bind labelled DNA at 30  C for 30 minutes in buffer A and typically 100 mM ATP. ([ATP] between 0.1 mM and 100 mM gave identical results, presumably because ATP was always in substantial excess over ligase or DNA.) T4 DNA ligase diluted in buffer A was added and the ligation performed at room temperature. Final ligase concentration ranged from 5-12,000 NEB units/ml depending on the J factor; [ligase] was adjusted to give substantial but incomplete ligation in 30 minutes. Eight 4 ml time points at 0, 1, 2, 3, 5, 15, 30, and 120 minutes were quenched by addition to 2 ml of 2 mg/ml proteinase K, 15 % glycerol, 75 mM EDTA and dyes. Reactions were heated for ten minutes

262 at 55  C and analyzed on 6 % native gels (75:1 acrylamide:bis, 90 mM Tris-borate, 1 mM EDTA, 0.8 mm thick, 20 cm long, run at 2.5 V/cm) containing 0.1 mg/ml ethidium bromide to resolve topoisomers. Dried gels were quanti®ed on a PhosphorImager. Data analysis The J factor is k1/k2, where k1 is the cyclization rate constant and k2 is the bimolecular ligation rate constant. The time dependence of the concentrations of the circular and bimolecular DNA were ®t to the expressions below, describing competing cyclization and bimolecular ligation (Crothers et al., 1992; Kahn & Crothers, 1992): P ( ) 1 ‡ 4‰MŠ0 …1 ÿ eÿ i k1;i t †k2 P ‰Ci Št ˆ …k1;i =k2 † ln …1† i k1;i P ( ) X 1 ‰MŠ0 eÿ i k1;i t P ‰Ci Št ÿ ‰BŠt ÿ ‰MŠ0 ˆ P 2 1 ‡ 4‰MŠ0 …1 ÿ eÿ i k1;i t †k2 = i k1;i i …2† where [Ci]t and [B]t are the concentrations of monomolecular circle i (when all circles originate from the same pool of starting material) and total bimolecular products, respectively, at time t, [M]0 is the initial concentration of ligateable DNA, k1,i is the cyclization rate constant for circle i, k2 is the intrinsic bimolecular rate constant and 4k2 is the apparent bimolecular rate constant of DNA with two ligateable ends (Taylor & Hagerman, 1990). [M]0 was estimated from the extent of reaction at two hours and from the curve ®tting; we routinely observed that the extent of reaction at two hours was greater than was predicted by the shorter time points, due to slow ligation processes described below. Curve ®tting was done using Kaleidagraph (Synergy Software) or MATLAB (The Mathworks, Inc.). Rate constants were normalized by the ligase concentration. We veri®ed that under our conditions the bimolecular rate constant is linearly dependent (within a factor of two) on ligase concentration at greater than 1000 units/ml ligase and that the cyclization rate constant is linearly dependent down to 20 units/ml (data not shown). For molecules which cyclize rapidly and where the bimolecular reaction is suppressed, J was calculated using the normalized cyclization rate constant and an average bimolecular rate constant derived from slowly cyclizing molecules analyzed at the same time, for which [ligase] > 1000 units/ml ligase was used. The normalized bimolecular rate constant varied no more than fourfold for experiments done on different days, and it was unaffected by TBP. The occupancy of the TATA box by TBP is typically about 30 %. Occupancy can be determined from cyclization data if TBP-bound DNA cyclizes to give a different topoisomer than free DNA, and in this case the data was modelled to give accurate estimates for cyclization rate constants (and therefore J factors) for each population. The kinetic equations, however, cannot be integrated because of cross terms arising from bimolecular ligation of bound and free DNA, so time courses are simulated by numerical integration using MATLAB, and best-®t kinetic constants are obtained by minimizing the error versus experiment. Occupancy was estimated in this way whenever new topoisomers appeared. TBP apparently does not exchange rapidly enough to average the cyclization rate;

TATA Box DNA Deformation with and without TBP for example, Figure 4 illustrates independent time courses for TBP reactions in one tube. When TBP-bound DNA gives the same DNA product (topoisomer) as the free DNA, it is more dif®cult to determine the J factors for each of the two populations, even when binding leads to much faster or slower cyclization. The observed biphasic kinetics usually do not allow simultaneous determination of TBP occupancy (amplitudes) and the independent rate constants, as defective molecules (discussed below) also contribute to the slow phase. The consequences of fractional occupancy are that the measured J factors are lower bounds when TBP enhances cyclization, and upper bounds when TBP represses cyclization. The ®tting procedures, which treat the active DNA concentration as an adjustable parameter, will tend to give results dominated by the fastest rates. Since the measured occupancy is typically >25 % when TBP enhances cyclization the error in J is no more than fourfold, and is probably signi®cantly less, as ®tting to a decreased apparent active DNA concentration will give an accurate rate constant for the bound population (the ``active'' DNA as far as the ®tting is concerned is the rapidly cyclizing bound form). However, when TBP represses cyclization, the apparent rate is dominated by the fast free DNA population, so the estimates for the cyclization of the bound DNA reported in Table 1 are only upper bounds. Non-ligateable or slowly ligateable defective DNA in the reaction probably originates from the ClaI digestion used to prepare cyclization substrates (DNA isolated from plasmids versus prepared by PCR shows a similar ``dead fraction''; data not shown). This defective DNA apparently can undergo very slow cyclization, giving a slow second phase for the appearance of circles for rapidly cyclizing molecules. (The slow ligation of defective ends is balanced by the high effective end concentration.) Bimolecular rate constants obtained at less than 1000 units/ml ligase are unreliable because they are due to defective molecules: if cyclization is rapid enough to require using low ligase concentration, then it is rapid enough so that there should be very little bimolecular reaction. This in¯ates estimates for k2, since the ®tting procedures will impose a large k2 in order to compete with rapid cyclization. For molecules with low J factors, the slow ligation of defective molecules adds only a small increment to the amount of observed bimolecular product. The derived bimolecular rate constant from these molecules is then assumed for rapidly cyclizing molecules analyzed at the same time, appropriately scaled by the ligase concentration.

Acknowledgements We are grateful to M. Brenowitz for yTBP, to S. Burley for aTBP, and to both for advice. Discussions with J. Maddocks and R. Manning led to the perpendicular bend model. The work was supported by a grant to J.D.K. from the National Institutes of Health (GM-53620), as well as startup funds and a Graduate Research Board award from the University of Maryland. N.D. was supported by a Patricia Roberts Harris fellowship from the University of Maryland.

TATA Box DNA Deformation with and without TBP

References Bates, A. D. & Maxwell, A. (1993). DNA Topology: In Focus (Rickwood, D. & Male, D., eds), IRL Press at Oxford University Press, Oxford. Bolshoy, A., McNamara, P., Harrington, R. E. & Trifonov, E. N. (1991). Curved DNA without A-A: experimental estimation of all 16 DNA wedge angles. Proc. Natl Acad. Sci. USA, 88, 2312-2316. Burley, S. K. & Roeder, R. G. (1996). Biochemistry and structural biology of transcription factor IID (TFIID). Annu. Rev. Biochem. 65, 769-799. Coleman, R. A. & Pugh, B. F. (1995). Evidence for functional binding and stable sliding of the TATA binding protein on nonspeci®c DNA. J. Biol. Chem. 270, 13850-13859. Coleman, R. A., Taggart, A. K. P., Benjamin, L. R. & Pugh, B. F. (1995). Dimerization of the TATA binding protein. J. Biol. Chem. 270, 13842-13849. Cox, J. M., Hayward, M. M., Sanchez, J. R., Cegnas, L. D., van der Zee, S., Dennis, J. H., Sigler, P. B. & Schepartz, A. (1997). Bidirectional binding of the TATA box binding protein to the TATA box. Proc. Natl Acad. Sci. USA, 94, 13475-13480. Crothers, D. M. & Steitz, T. A. (1992). Transcriptional activation by Escherichia coli CAP protein. In Transcriptional Regulation (McKnight, S. L. & Yamamoto, K. R., eds), vol. 1, pp. 501-534, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Crothers, D. M., Drak, J., Kahn, J. D. & Levene, S. D. (1992). DNA bending, ¯exibility, and helical repeat by cyclization kinetics. Methods Enzymol. 212, 1-29. De Santis, P., FuaÁ, M., Savino, M., Anselmi, C. & Bocchinfuso, G. (1996). Sequence dependent circularization of DNAs: a physical model to predict the DNA sequence dependent propensity to circularization and its changes in the presence of proteininduced bending. J. Phys. Chem. 100, 9968-9976. Diagana, T. T., North, D. L., Jabet, C., Fiszman, M. Y., Takeda, S. & Whalen, R. G. (1997). The transcriptional activity of a muscle-speci®c promoter depends critically on the structure of the TATA element and its binding protein. J. Mol. Biol. 265, 480-493. Dripps, D. & Wartell, R. M. (1987). DNA bending induced by the catabolite activator protein allows ring formation of a 144 bp DNA. J. Biomol. Struct. Dynam. 5, 1-13. Flatters, D., Young, M., Beveridge, D. L. & Lavery, R. (1997). Conformational properties of the TATA-box binding sequence of DNA. J. Biomol. Struct. Dynam. 14, 757-765. Flory, P. J., Suter, U. W. & Mutter, M. (1976). Macrocyclization equilibria. I. theory. J. Am. Chem. Soc. 98, 5733-5739. Frank-Kamenetskii, M. D. (1990). DNA supercoiling and unusual structures. In DNA Topology and Its Biological Effects (Cozzarelli, N. R. & Wang, J. C., eds), pp. 185-215, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Geiger, J. H., Hahn, S., Lee, S. & Sigler, P. B. (1996). Crystal structure of the yeast TFIIA/TBP/DNA complex. Science, 272, 830-836. Grosschedl, R. (1995). Higher-order nucleoprotein complexes in transcription: analogies with site-speci®c recombination. Curr. Opin. Cell Biol. 7, 362-370. Grove, A., Galeone, A., Yu, R., Mayol, L. & Geiduschek, E. P. (1998). Af®nity, stability and polarity of bind-

263 ing of the TATA binding protein governed by ¯exure at the TATA box. J. Mol. Biol. 282, 731-739. Guzikevich-Guerstein, G. & Shakked, Z. (1996). A novel form of the DNA double helix imposed on the TATA-box by the TATA-binding protein. Nature Struct. Biol. 3, 32-37. Hagerman, P. J. (1996). Do basic region-leucine zipper proteins bend their DNA targets ... does it matter? Proc. Natl Acad. Sci. USA, 93, 9993-9996. Hagerman, P. J. & Ramadevi, V. A. (1990). Application of the method of phage T4 DNA ligase-catalyzed ring-closure to the study of DNA structure. I. Computational analysis. J. Mol. Biol. 212, 351-362. Hahn, S., Buratowski, S., Sharp, P. A. & Guarente, L. (1989). Yeast TATA-binding protein TFIID binds to TATA elements with both consensus and nonconsensus DNA sequences. Proc. Natl Acad. Sci. USA, 86, 5718-5722. Haran, T. E., Kahn, J. D. & Crothers, D. M. (1994). Sequence elements responsible for DNA curvature. J. Mol. Biol. 244, 135-143. Harrington, R. E. (1993). Studies of DNA bending and ¯exibility using gel-electrophoresis. Electrophoresis, 14, 732-746. Hernandez, N. (1993). TBP, a universal eukaryotic transcription factor? Genes Dev. 7, 1291-1308. Hockings, S. C., Kahn, J. D. & Crothers, D. M. (1998). Recognition of the AP-1 and ATF/CREB sites by GCN4. Proc. Natl Acad. Sci. USA, 95, 1410-1415. Hoopes, B. C., LeBlanc, J. F. & Hawley, D. K. (1992). Kinetic analysis of yeast TFIID-TATA box complex formation suggests a multi-step pathway. J. Biol. Chem. 267, 11539-11547. Hoopes, B. C., LeBlanc, J. F. & Hawley, D. K. (1998). Contributions of the TATA box sequence to ratelimiting steps in transcription initiation by RNA polymerase II. J. Mol. Biol. 277, 1015-1031. Jacobson, H. & Stockmayer, W. H. (1950). Intramolecular reaction in polycondensations. I. The theory of linear systems. J. Chem. Phys. 18, 1600-1606. Juo, Z. S., Chiu, T. K., Lieberman, P. M., Baikalov, L., Berk, A. J. & Dickerson, R. E. (1996). How proteins recognize the TATA box. J. Mol. Biol. 261, 238-254. Kahn, J. D. & Crothers, D. M. (1992). Protein-induced bending and DNA cyclization. Proc. Natl Acad. Sci. USA, 89, 6343-6347. Kahn, J. D. & Crothers, D. M. (1993). DNA bending in transcription initiation. Cold Spring Harbor Symp. Quant. Biol. 58, 115-122. Kahn, J. D. & Crothers, D. M. (1998). Measurement of the DNA bend angle induced by the catabolite activator protein using Monte Carlo simulation of cyclization kinetics. J. Mol. Biol. 276, 287-309. Kahn, J. D., Yun, E. & Crothers, D. M. (1994). Detection of localized DNA ¯exibility. Nature, 368, 163-166. Kerppola, T. K. (1996). Fos and Jun bend the AP-1 site: effects of probe geometry on the detection of protein-induced DNA bending. Proc. Natl Acad. Sci. USA, 93, 10117-10122. Ê resolution re®ned Kim, J. L. & Burley, S. K. (1994). 1.9 A structure of TBP recognizing the minor groove of TATAAAAG. Nature Struct. Biol. 1, 638-653. Kim, J. L., Nikolov, D. B. & Burley, S. K. (1993a). Cocrystal structure of TBP recognizing the minor groove of a TATA element. Nature, 365, 520-527. Kim, Y., Geiger, J. H., Hahn, S. & Sigler, P. B. (1993b). Crystal structure of a yeast TBP/TATA-box complex. Nature, 365, 512-520.

264 Koo, H.-S., Drak, J., Rice, J. A. & Crothers, D. M. (1990). Determination of the extent of DNA bending by an adenine-thymine tract. Biochemistry, 29, 4227-4234. Kotlarz, D., Fritsch, A. & Buc, H. (1986). Variations of intramolecular ligation rates allow the detection of protein-induced bends in DNA. EMBO J. 5, 799-803. Kuprash, D. V., Rice, N. R. & Nedospasov, S. A. (1995). Homodimer of p50 (NFkB1) does not introduce a substantial directed bend into DNA according to three different experimental assays. Nucl. Acids Res. 23, 427-433. Laundon, C. H. & Grif®th, J. D. (1988). Curved helix segments can uniquely orient the topology of supertwisted DNA. Cell, 52, 545-549. Lebrun, A., Shakked, Z. & Lavery, R. (1997). Local DNA stretching mimics the distortion caused by the TATA box-binding protein. Proc. Natl Acad. Sci. USA, 94, 2993-2998. Lorch, Y. & Kornberg, R. D. (1993). Near-zero linking difference upon transcription factor IID binding to promoter DNA. Mol. Cell. Biol. 13, 1872-1875. Manning, R. S., Maddocks, J. H. & Kahn, J. D. (1996). A continuum rod model of sequence-dependent DNA structure. J. Chem. Phys. 105, 5626-5646. Marko, J. F. & Siggia, E. D. (1994). Bending and twisting elasticity of DNA. Macromolecules, 27, 981-988. McCormick, R. J., Badalina, T. & Fisher, D. E. (1996). The leucine zipper may induce electrophoretic mobility anomalies without DNA bending. Proc. Natl Acad. Sci. USA, 93, 14434-14439. Miaskiewicz, K. & Ornstein, R. L. (1996). DNA binding by TATA-box binding protein (TBP): a molecular dynamics computational study. J. Biomol. Struct. Dynam. 13, 593-600. Mizutani, M., Ohta, T., Watanabe, H., Handa, H. & Hirose, S. (1991). Negative supercoiling of DNA facilitates an interaction between transcription factor IID and the ®broin gene promoter. Proc. Natl Acad. Sci. USA, 88, 718-722. Nikolov, D. B., Chen, H., Halay, E. D., Usheva, A. A., Hisatake, K., Lee, D. K., Roeder, R. G. & Burley, S. K. (1995). Crystal structure of a TFIIB-TBP-TATA element ternary complex. Nature, 377, 119-128. Nikolov, D. B., Chen, H., Halay, E. D., Hoffman, A., Roeder, R. G. & Burley, S. K. (1996). Crystal structure of a human TATA box-binding protein/TATA element complex. Proc. Natl Acad. Sci. USA, 93, 4862-4867. OelgeschlaÈger, T., Chiang, C.-M. & Roeder, R. (1996). Topology and reorganization of a human TFIIDpromoter complex. Nature, 382, 735-738. Paolella, D. N., Palmer, C. R. & Schepartz, A. (1994). DNA targets for certain bZIP proteins distinguished by an intrinsic bend. Science, 264, 1130-1133. Pardo, L., Pastor, N. & Weinstein, H. (1998). Progressive DNA bending is made possible by gradual changes in the torsion angle of the glycosyl bond. Biophys. J. 74, 2191-2198. Parkhurst, K., Brenowitz, M. & Parkhurst, L. J. (1996). Simultaneous binding and bending of promoter DNA by the TATA binding protein: real time kinetic measurements. Biochemistry, 35, 7459-7465. Parvin, J. D., McCormick, R. J., Sharp, P. A. & Fisher, D. E. (1995). Pre-bending of a promoter sequence enhances af®nity for the TATA-binding factor. Nature, 373, 724-727. Pastor, N., Pardo, L. & Weinstein, H. (1997). Does TATA matter? A structural explanation of the selec-

TATA Box DNA Deformation with and without TBP tivity determinants in its complexes with TATA box-binding protein. Biophys. J. 73, 640-652. Patel, D. J., Kozlowski, S. A., Weiss, M. & Matt, R. (1985). Conformation and dynamics of the Pribnow box region of the self-complementary d(C-C-A-T-TA-T-A-A-T-C-G) duplex in solution. Biochemistry, 1985, 936-944. Pellegrini, L., Tan, S. & Richmond, T. J. (1995). Structure of serum response factor core bound to DNA. Nature, 376, 490-498. Perez-Howard, G. M., Weil, P. A. & Beechem, J. M. (1995). Yeast TATA binding protein interaction with DNA: ¯uorescence determination of oligomeric state, equilibrium binding, on-rate, and dissociation kinetics. Biochemistry, 34, 8005-8017. Petri, V., Hsieh, M. & Brenowitz, M. (1995). Thermodynamic and kinetic characterization of the binding of the TATA binding protein to the adenovirus E4 promoter. Biochemistry, 34, 9977-9984. Robert, E., Douziech, M., Forget, D., Egly, J.-M., Greenblatt, J., Burton, Z. & Coulombe, B. (1998). Wrapping of promoter DNA around the RNA polymerase II initiation complex induced by TFIIF. Mol. Cell, 2, 341-351. Ryu, S., Garges, S. & Adhya, S. (1994). An arcane role of DNA in transcription activation. Proc. Natl Acad. Sci. USA, 91, 8582-8586. Santero, E., Hoover, T. R., North, A. K., Berger, D. K., Porter, S. C. & Kustu, S. (1992). Role of integration host factor in stimulating transcription from the s54-dependent nifH promoter. J. Mol. Biol. 227, 602620. Schultz, S. C., Shields, C. C. & Steitz, T. A. (1991). Crystal structure of a CAP-DNA complex: the DNA is bent by 90  . Science, 253, 1001-1007. Schurr, J. M., Delrow, J. J., Fujimoto, B. S. & Benight, A. S. (1997). The question of long-range allosteric transitions in DNA. Biopolymers, 44, 283-308. Shore, D. & Baldwin, R. L. (1983a). Energetics of DNA twisting: I. Relation between twist and cyclization probability. J. Mol. Biol. 170, 957-981. Shore, D. & Baldwin, R. L. (1983b). Energetics of DNA twisting: II. Topoisomer analysis. J. Mol. Biol. 170, 983-1007. Shore, D., Langowski, J. & Baldwin, R. L. (1981). DNA ¯exibility studied by covalent closure of short fragments into circles. Proc. Natl Acad. Sci. USA, 78, 4833-4837. Sitlani, A. & Crothers, D. M. (1996). Fos and Jun do not bend the AP-1 recognition site. Proc. Natl Acad. Sci. USA, 93, 3248-3252. Sitlani, A. & Crothers, D. M. (1998). DNA-binding domains of Fos and Jun do not induce DNA curvature: an investigation with solution and gel methods. Proc. Natl Acad. Sci. USA, 95, 1404-1409. Starr, D. B., Hoopes, B. C. & Hawley, D. K. (1995). DNA bending is an important component of site-speci®c recognition by the TATA binding protein. J. Mol. Biol. 250, 434-446. Suzuki, M. & Yagi, N. (1995). Stereochemical basis of DNA bending by transcription factors. Nucl. Acids Res. 23, 2083-2091. Tabuchi, H., Handa, H. & Hirose, S. (1993). Underwinding of DNA on binding of yeast TFIID to the TATA element. Biochem. Biophys. Res. Commun. 192, 14321438. Tan, S., Hunziker, Y., Sargent, D. F. & Richmond, T. J. (1996). Crystal structure of a yeast TFIIA/TBP/ DNA complex. Nature, 381, 127-134.

TATA Box DNA Deformation with and without TBP Tang, H., Sun, X., Reinberg, D. & Ebright, R. H. (1996). Protein-protein interactions in eukaryotic transcription initiation: structure of the preinitiation complex. Proc. Natl Acad. Sci. USA, 93, 1119-1124. Taylor, W. H. & Hagerman, P. J. (1990). Application of the method of phage T4 DNA ligase-catalyzed ringclosure to the study of DNA structure. II. Sodium chloride-dependence of DNA ¯exibility and helical repeat. J. Mol. Biol. 212, 363-376. TenHarmsel, A. & Biggin, M. D. (1995). Bending DNA can repress a eukaryotic basal promoter and inhibit TFIID binding. Mol. Cell. Biol. 15, 5492-5498.

265 Ulanovsky, L., Bodner, M., Trifonov, E. N. & Choder, M. (1986). Curved DNA: design, synthesis and circularization. Proc. Natl Acad. Sci. USA, 83, 862-866. Widlund, H. R., Cao, H., Simonsson, S., Magnusson, E., Simonsson, T., Nielsen, P. E., Kahn, J. D., Crothers, D. M. & Kubista, M. (1997). Identi®cation and characterization of genomic nucleosome-positioning sequences. J. Mol. Biol. 267, 807-817. Wobbe, C. R. & Struhl, K. (1990). Yeast and human TATA-binding proteins have nearly identical DNA sequence requirements for transcription in vitro. Mol. Cell. Biol. 10, 3859-3867. Zinkel, S. S. & Crothers, D. M. (1987). DNA bend direction by phase sensitive detection. Nature, 328, 178-181.

Edited by R. Ebright (Received 30 November 1998; received in revised form 1 June 1999; accepted 1 June 1999)