(Received for publication, May 5, 1995; and in revised form, June 9, 1995)
From the
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 -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
M
for major late promoter DNA,
whereas the second binding event occurs with a remarkably reduced
affinity, K
= (1.2 ± 0.8)
10
M
. 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.
The human transcription factor USF ()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/HLH
DNA 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
-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.
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 F versus time was fitted to a number of nonlinear analytical equations
using Peakfit software from Jandel Scientific.
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. F was calculated at 335 nm where
F = F
- F
.
The K of b/HLH
oligonucleotide and
b/HLH/Z
oligonucleotide 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
M
for the interaction of b/HLH with
MLP oligonucleotide.
Figure 2:
1 µ M b/HLH dimer fluorescence
quenching by 0-10 µM double-stranded MLP (),
LCR (
), and NS DNA (
).
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.
Figure 4:
Single exponential curve fitting of the
F 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
F
is the maximum
fluorescence change.
where k 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
[C] + k
.
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 = (k
)/k
. rearranges to
A plot of 1/k versus
1/[D] will give an intercept of 1/k
(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
was obtained as the
reciprocal of y intercept (134.4 ± 9.8
s
).
This simple model gives a k 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.
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/HLHDNA 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
(Table 2, k
=
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.
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
DNAUSF 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 USFDNA
complex is relatively easy and allows reconstruction of the
USF
looped 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.