©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Anti-cooperative Biphasic Equilibrium Binding of Transcription Factor Upstream Stimulatory Factor to Its Cognate DNA Monitored by Protein Fluorescence Changes (*)

(Received for publication, May 5, 1995; and in revised form, June 9, 1995)

Ma Sha (1) Adrian R. Ferré-D'Amaré (2)(§) Stephen K. Burley (2)(¶) Dixie J. Goss (1)(**)

From the  (1)Department of Chemistry, Hunter College of the City University of New York, New York, New York 10021-5024 and the (2)Laboratories of Molecular Biophysics, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Upstream stimulatory factor USF is a human transcriptional activation factor, which uses a basic/helix-loop-helix/leucine zipper (b/HLH/Z) motif to homodimerize and recognize specific sequences in the promoter region of both nuclear and viral genes transcribed by RNA polymerase II. Steady state fluorescence spectroscopy demonstrated that the basic/helix-loop-helix/leucine zipper domain of USF binds its DNA targets with high affinity and specificity, whereas removal of the leucine zipper yielding the basic/helix-loop-helix minimal DNA binding region reduces both affinity and specificity. Stopped flow method provided kinetic evidence for a two-step binding process involving rapid formation of a protein-DNA intermediate followed by a slow isomerization step, which is consistent with the basic region undergoing a random coil to alpha-helix folding transition on specific DNA recognition. The leucine zipper is also necessary for USF to function as a bivalent homotetramer, capable of binding two distinct recognition sites simultaneously and mediating DNA looping under physiologic conditions. Titration studies revealed that the first binding event has a equilibrium constant K = (2.2 ± 2.0) 10^9M for major late promoter DNA, whereas the second binding event occurs with a remarkably reduced affinity, K = (1.2 ± 0.8) 10^8M. This anticooperative feature of DNA binding by the homotetramer suggests that USF stimulates transcription by mediating DNA looping between nearby recognition sites located in class II nuclear and viral gene promoters.


INTRODUCTION

The human transcription factor USF (^1)is involved in the regulation of both cellular and viral genes. USF belongs to the basic (b)/helix-loop-helix (HLH)/leucine zipper (Z) family of transcription factors and stimulates transcription by binding to a specific E-box motif (CACGTG) upstream of many cellular and viral promoters. Earlier studies identified USF as a cellular factor that activates the intrinsically strong adenovirus major late promoter by binding to an E-box located at position -60 with respect to the cap site (Carthew et al., 1985; Miyamoto et al., 1985; Sawadogo and Roeder, 1985) and stimulates transcription, possibly by direct interaction with the transcription factor TFIID (Sawadogo et al., 1988; Meisterernst et al., 1985; Bungert et al., 1992). USF contains four major domains: A, activation; b, basic domain; HLH, helix-loop-helix domain, and Z, leucine zipper domain. The leucine zipper domain was believed to be necessary for high affinity DNA binding by b/HLH/Z (Ferré-D'Amaréet al., 1994). The present studies used the b/HLH and b/HLH/Z domains of USF. The recent cocrystal structure of b/HLHbulletDNA has shown that the b/HLH binds to one duplex DNA as a dimer, and other studies suggested that the b/HLH/Z bind two duplex DNAs at the same time as a homotetramer (Ferré-D'Amaréet al., 1994), but little is known about the binding affinity, specificity, or the rate of this reaction. The duplex DNA oligomer derived from the adenovirus MLP promoter containing a palindromic E-box (underlined) was used in this study.

A nonspecific (NS) sequence, which contains three mutations (labeled with *) on one half of the sequence, was designed to alter the E-box.

Recent studies showed that USF is a component of human beta-globin locus control region (LCR) transcription complex with a binding site very much like our NS sequence (Bresnick and Felsenfeld, 1993).

The comparison of the association constants of USF with LCR, NS and the intrinsically strong MLP sequence will shed light on the sequence-specific interaction of USF with DNA.


MATERIALS AND METHODS

DNA Oligomers and Protein

USF DNA binding site oligomers of MLP, LCR, and NS were synthesized, purified, quantitated and annealed as described elsewhere (Ferré-D'Amaréet al., 1993). The overexpression and purification of both protein constructs has been described previously (Ferré-D'Amaréet al., 1994). Cysteine-free mutants, which are indistinguishable from wild-type protein, were employed.

Fluorescence Measurements

The binding buffer used for fluorescence measurements consisted of 10 mM HEPES-KOH, 100 mM KCl, 10% glycerol, and 1 mM MgCl(2), adjusted to pH 7.5. All chemicals were reagent grade or better. Fluorescence measurements were carried out at 25 °C, unless otherwise noted, on a SPEX Fluorolog-2 spectrofluorometer equipped with a high intensity xenon arc lamp, and steady state data were collected and analyzed as described previously in detail (Sha et al., 1994; Carberry et al., 1989, 1990).

The kinetics of the binding of the duplex E-box sequence of MLP DNA to the b/HLH and b/HLH/Z domains of USF were followed by measuring the changes of the intrinsic protein fluorescence after rapid mixing by a Hi-Tech Scientific SFA-11 stopped-flow apparatus. The excitation wavelength was 281 nm, and the slit width was 2 mm. Light emitted from the reaction mixture was monitored at a single wavelength, 335 nm, with a integration time of 1 ms. A series of stopped-flow experiments were performed at different temperatures. After rapid mixing of protein (0.1 µM b/HLH dimer or 0.1 µM b/HLH/Z tetramer) with the appropriate DNA solution, the time course of the intrinsic fluorescence intensity was recorded. The plot of DeltaF versus time was fitted to a number of nonlinear analytical equations using Peakfit software from Jandel Scientific.


RESULTS

Steady State Fluorescence Measurements

The fluorescence emission spectra of b/HLHbulletoligonucleotide complexes as a function of oligonucleotide concentration is shown in Fig. 1. Upon complex formation, there is a decrease in the protein fluorescence intensity at 335 nm. Such protein fluorescence quenching has been attributed to the - stacking interactions between an aromatic amino acid residue, in this case tryptophan (Trp-218) and the nucleic acid base (Lawaczek and Wagner, 1974; Brun et al., 1975; Ishida et al., 1983).


Figure 1: Fluorescence emission spectra of b/HLH domain of USF (0.2 µM dimer) titrated with double-stranded MLP oligonucleotide in the binding buffer, pH 7.5, at 25 °C. The oligonucleotide concentration (top to bottom) was 0.0, 0.2, 0.8, 1.8, 2.8, 4.0, and 6.0 µM. The excitation wavelength was 281 nm, and a 1.4-mm slit was employed. Emission maxima was observed at 335 nm. Inset, Eadie-Hofstee plot of fluorescence data. DeltaF was calculated at 335 nm where DeltaF = F - F.



The K of b/HLHbulletoligonucleotide and b/HLH/Zbulletoligonucleotide complex formation can be calculated from the relative fluorescence intensity changes in free and complexed protein emission spectra by construction of an Eadie-Hofstee plot, as shown in the inset of Fig. 1. K was found to be (4.7 ± 0.6) 10^5M for the interaction of b/HLH with MLP oligonucleotide.

Quenching of b/HLH Fluorescence by DNA

The recent cocrystal structure of b/HLHbulletDNA has shown that b/HLH binds to one duplex DNA as a dimer (Ferré-D'Amaréet al., 1994). We used concentration of dimer as the protein concentration in fitting the fluorescence titration data. The binding of MLP, LCR, and NS DNA to b/HLH showed similar simple quenching curves (Fig. 2). All three DNA bind b/HLH with about the same affinity (Table 1, MLP, K = (4.7 ± 0.6) 10^5M; LCR, K = (4.0 ± 0.2) 10^5M; NS, (3.2 ± 0.1) 10^5M). The specificity of binding is defined as the equilibrium constant for binding to specific DNA sequences divided by the binding equilibrium constant for binding to nonspecific DNA. Thus, the binding specificity of b/HLH for MLP E-box duplex oligonucleotide compared with the NS sequence is only 1.47, and the binding specificity of b/HLH for LCR binding site oligonucleotide compared with the NS sequence is only 1.25, which suggests little specificity of binding for either oligonucleotide (Table 1). This is in dramatic contrast to the b/HLH/Z protein results shown below.


Figure 2: 1 µ M b/HLH dimer fluorescence quenching by 0-10 µM double-stranded MLP (), LCR (), and NS DNA ().





Biphasic Binding of b/HLH/Z with DNA

b/HLH/Z forms a homotetrameric complex that has been shown to bind two DNA duplex molecules at the same time (Ferré-D'Amaréet al., 1994). We used a stoichiometry of four b/HLH/Z monomer/two DNA duplex for analysis of the fluorescence titrations. The binding of MLP and LCR DNA to b/HLH/Z showed a dramatic biphasic binding mode (enhancement of fluorescence followed by fluorescence quenching (Fig. 3, A and B)), while the binding of the NS sequence to b/HLH/Z showed a single binding mode (Fig. 3C) as seen for the b/HLH protein. For the biphasic binding of b/HLH/Z to MLP and LCR, the turning point from fluorescence enhancement to fluorescence quenching was at a 1:1 ratio of tetramer b/HLH/Z to duplex DNA (Fig. 3A, 50 nM b/HLH/Z tetramer, 50 nM DNA duplex; Fig. 3B, 0.5 µM b/HLH/Z tetramer, 0.5 µM DNA duplex). Which clearly indicates that two DNAs are binding to a single tetrameric b/HLH/Z and that the two DNA binding sites on the same protein tetramer are not equivalent. The first DNA molecule binding induced a fluorescence enhancement, while the second DNA molecule binding induced fluorescence quenching. b/HLH/Z showed very specific binding toward MLP DNA. The binding constant of b/HLH/Z for MLP is about 1,000-10,000-fold stronger than the binding of the other two DNA (Table 1; MLP, first binding mode, (2.2 ± 2.0) 10^9M; second binding mode, (1.2 ± 0.8) 10^8M; LCR, first binding mode, (3.1 ± 3.7) 10^6M; second binding mode, (3.3 ± 0.6) 10^5M; NS, second binding mode only, (1.1 ± 0.3) 10^5M). The specificity of b/HLH/Z binding to MLP E-box compared with the NS sequence is 2.0 10^4 for the first and 1.1 10^3 for the second binding mode. The specificity of b/HLH/Z binding to the LCR sequence compared with the NS sequence is 28 for the first and 3.0 for the second binding mode (Table 1). Results showed that b/HLH/Z has highly specific binding to the adenovirus major late promoter palindromic E-box sequence and moderately specific binding for human locus control region nonpalindrome E-box like sequence when compared with the nonspecific DNA sequence.


Figure 3: A, biphasic fluorescence changes of 50 nM tetramer b/HLH/Z upon binding of 0-400 nM MLP E-box sequence; B, biphasic fluorescence changes of 0.5 µM tetramer b/HLH/Z binding with LCR DNA; C, single-phase fluorescence changes of 0.5 µM tetramer b/HLH/Z binding with NS DNA.



Stopped Flow Fluorescence Kinetics

The stopped-flow data for the binding of MLP DNA to the b/HLH and b/HLH/Z were plotted as DeltaFversus time as shown in Fig. 4. Fitted curves correspond to the following single-exponential equation (Olsen et al., 1993).


Figure 4: Single exponential curve fitting of the DeltaF versus time for the kinetics of 0.1 µM b/HLH protein mixing with 1 µM MLP DNA. k is obtained from the fitted curve. Bottominset is the residue of fitting plotted on the same and but different y axis (rightsidebottom).



where k is the observed first-order rate constant, and the DeltaF is the maximum fluorescence change.

Mechanisms for MLP-b/HLH Interaction

Two possible schemes, a one-step reaction and a two-step reaction, for simple quenching of b/HLH by MLP E-box sequences were considered (Garland, 1978).

The one-step reaction is

where k(1) and k are forward and reverse rate constants, respectively; P an D refer to protein and DNA. Under the pseudo-first order condition, the observed rate constant is predicted to be a linear function of substrate concentration, i.e.k = k(1)[C] + k.

The two-step reaction is

which involves a fast association of protein and DNA followed by a slow change of conformation of the first association complex, PD*, to the stable complex, PD.

The interaction of b/HLH (0.1µM) with MLP E-box under different concentrations of DNA (1,2,and3.5µM) gave about the same k (125.8 ± 4.9, 128.9 ± 8.6, and 129.7 ± 5.9 s, respectively) within experimental error. The reaction is not a single-step pseudo-first order reaction (). Earlier CD experiments have shown that the b/HLH protein undergoes a 48% conformational change upon binding to MLP sequence (Ferré-D'Amaréet al., 1994), in agreement with . can be written as (Olsen et al., 1993)

where K(1) = (k)/k(1). rearranges to

If one assumes that k(2)k, then

and

A plot of 1/k versus 1/[D] will give an intercept of 1/k(2) (Fig. 5).


Figure 5: Kinetics plot of 1/kversus 1/[D] of 0.1 µM b/HLH under different MLP DNA concentrations (1, 2, 3.5, and 5 µM, respectively). k(2) was obtained as the reciprocal of y intercept (134.4 ± 9.8 s).



This simple model gives a k(2) value of 134.4 ± 9.8 s for b/HLH binding with MLP DNA. While more complex models with additional conformational changes will also fit the data, at present there is no experimental data to suggest that such a mechanism is required.

Mechanisms for MLP-b/HLH/Z Interaction

Four possible mechanisms that can be written for the biphasic (enhancement-quenching) binding behavior of MLP to b/HLH/Z are shown below.

Assuming that the b/HLH/Z protein complex is a symmetrical homotetramer, the two DNA binding sites would be equivalent prior to the first DNA binding. However, the two DNA binding sites on b/HLH/Z are dramatically different, one causes fluorescence enhancement, and the other causes fluorescence quenching. This can be explained as the result of the first DNA binding causing a conformational change on the second DNA binding site on b/HLH/Z. The conformational change on the first DNA binding event further exposes the tryptophan on the second binding site, thus giving tryptophan fluorescence enhancement instead of fluorescence quenching. The confirmation of conformational changes for first DNA binding rules out the possible and , which suggested one simple binding in the enhancement mode that does not contain a conformational change.

The stopped-flow data of b/HLH/Z interaction with MLP DNA was measured as previous described for b/HLH protein. The first DNA binding (fluorescence enhancement phase) for the kinetic interaction was too fast to be observed. We have been able to capture only the fluorescence change of the quenching phase (second DNA binding) of the b/HLH/Z DNA interaction. A simple relaxation curve resembling the curve obtained from b/HLHbulletDNA interaction was fitted by the same method as described earlier. The similar concentration independence of k, 82.4 ± 7.2, 84.5 ± 8.9, and 85.4 ± 4.5 s, at different DNA concentrations respectively (Table 2), suggested the involvement of a conformational change as discussed in detail for b/HLH DNA interaction. The initial binding of second DNA is much faster than the conformational change step that follows. The observed k approximately equals to the calculated k(4) (Table 2, k(4) = 87.5 ± 10.6 s), the rate constant for the last step (conformational change) in b/HLH/Z MLP binding. The only mechanism that contains two conformational changes is for b/HLH/Z binding with MLP DNA. This mechanism is consistent with the observed kinetics.




DISCUSSION

Although the leucine zipper motif is not involved in direct contact with DNA during b/HLH/Z binding, it has some crucial effects on DNA binding to the protein. The fact that b/HLH/Z had 1000-100-fold (first DNA binding and second DNA binding, respectively) higher affinity to MLP than b/HLH suggests that the leucine zipper is necessary for high affinity DNA binding. Deletion of the leucine zipper causes the protein to lose the ability to discriminate among DNA sequences (Table 1). Presumably the leucine zipper motif maintains tetramerization of the protein that seems to be important both in binding affinity and in specificity.

Both LCR and NS DNA have a significantly lower affinity for b/HLH/Z compared with MLP DNA (Table 1). However, LCR DNA still exhibits a biphasic interaction (enhancement, then quenching), while the NS DNA has only one quenching mode. Although no longer a palindrome, the LCR sequence maintained a less distorted E-box than that of the NS oligonucleotide, which has an extra mutation next to the E-box.

These binding affinity results demonstrated that the base immediately outside the E-box has some effect on specific binding especially on a slightly modified E-box sequence. The palindrome in the E-box is a crucial factor for high affinity binding, even a one-base substitution that disturbed this symmetry (from MLP to LCR or NS) caused at least a 100-fold affinity decrease (Table 1).

It was demonstrated earlier that the b/HLH/Z tetramer binds two DNA simultaneously (Ferré-D'Amaréet al., 1994). This study shows a sequential binding of two DNA molecules to b/HLH/Z that exhibits an unusual negative cooperativity. A 10-fold decrease on the second DNA binding affinity was observed for both the MLP and the LCR sequences. An anti-cooperative binding mechanism, although unusual, will benefit DNA looping and mismatch correction in the transcription system as explained below (Fig. 6). DNA looping for USF was suggested earlier (Ferré-D'Amaréet al., 1994). DNA looping by USF requires two E-box sequences on the same DNA functioning together. Double E-box sequences are found in many systems. Two equivalent E-box sequences, the insulin enhancer binding sequences (IEB1 and IEB2) were found in the human insulin gene enhancer that binds a helix-loop-helix transcription factor, insulin enhancer factor IEF1. At least one of the IEB sequences was found to bind USF (Read et al., 1993). The in vivo transcriptional activity of the insulin gene was only modestly affected (less than 2-fold) when the distance between the two E-box (IEB1 and IEB2) elements are changed in half-integral number of double-helical turns, and the introduction of more than two E-boxes sharply reduce the activity (Leshkowitz et al., 1992). Two copies of LCR sequences in mice were found binding to USF and functioning together. No activity was observed when there is only one LCR (Ellis et al., 1993). Paired E-box sequences were found necessary in mouse muscle creatin kinase gene enhancer, which loses activity when one E-box is removed (Martin et al., 1994).


Figure 6: Model of selectivity for looped DNAbulletUSF complex formation by anti-cooperative binding during transcription activation.



Although USF in different systems may stimulate transcription in a variety of ways, many studies have suggested that in some double E-boxed transcription systems, USF stimulate transcription by DNA looping. In a system where DNA looping is necessary for transcription activation, the anticooperative binding of E-box to USF measured in this study will actually benefit DNA looping and mismatch correction (a model is shown in Fig. 6). Because only looped DNA is activated, two DNA templates connected together by USF (mismatch) on their E-box will not function. However, the second DNA binding is 10 times less than the first one, therefore release of the second DNA from the USFbulletDNA complex is relatively easy and allows reconstruction of the USFbulletlooped DNA complex. The second E-box on the same DNA is generally closer and more available, i.e. has a much higher effective concentration compared with the E-box on another DNA strand. This negative cooperativity derived selectivity on looped DNA formation will benefit looping and enhance transcription activation.


FOOTNOTES

*
This work was supported by Grants MCB-9303661 and GER 9023681 from the National Science Foundation (to D. J. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a David Rockefeller predoctoral fellowship. Present address: Dept. of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114.

Investigator in the Howard Hughes Medical Institute.

**
To whom correspondence should be addressed: Dept. of Chemistry, Hunter College of the City University of New York, 695 Park Ave., New York, NY 10021-5024.

(^1)
The abbreviations used are: USF, upstream stimulating factor; b, basic; HLH, helix-loop-helix; Z, zipper; MLP, major late promoter; NS, nonspecific; LCR, locus control region; IEB, insulin enhancer binding.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.