(Received for publication, August 19, 1996, and in revised form, October 23, 1996)
From the Department of Pathology and Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262
The mammalian nucleus contains 6 billion base pairs of DNA, encoding about 100,000 genes, yet in a given cell steroid hormones induce only a handful of genes. The logistical difficulties faced by steroid receptors or other transcription factors of sorting through this much genetic information is further increased by the density of nuclear DNA (approximately 10-50 mg/ml). Standard models propose that steroid receptors find target elements by repeated cycles of dissociation and reassociation until a high affinity site is found (cycling model) and/or by conducting a one-dimensional search along the DNA (sliding model). A third model proposes that steroid receptors search for target sites in the genome by DNA intersegment transfer. In this model, receptor dimers bind nonspecific DNA sequences and search for a target site by binding a second strand of DNA before dissociating from the first, in effect moving through the genome like Tarzan swinging from vine to vine. This model has the advantage that a high concentration of DNA favors, rather than hinders, the search. The intersegment transfer model predicts, in contrast to the cycling and sliding models, that the dissociation rate of receptor from DNA is highly dependent on DNA concentration. We have employed the purified DNA binding domain fragment from the rat glucocorticoid receptor to perform equilibrium and kinetic studies of the DNA dependence of receptor-DNA dissociation. We find receptor dissociation from DNA to be highly dependent on the concentration of DNA in solution, in agreement with the intersegment transfer model. We also find that this interaction is primarily electrostatic, because DNA-like polyanion chains (e.g. heparin and polyglutamate) can mediate the transfer. These studies provide evidence that direct DNA transfer aids the target site search conducted by steroid receptors in their role as inducible transcription factors.
Many eukaryotic transcription factors function by binding to specific DNA sequences upstream of transcription start sites. In order to bind these DNA elements, the protein must first locate these sites in the genome. In humans this search entails locating a few functional binding sites from over 6 billion base pairs of DNA, requiring the protein to sample a vast number of possible binding sites in a very short period of time. Nevertheless, transcription factors are able to find their specific binding sites very rapidly. What are the mechanisms used to locate specific binding sites in chromatin?
One model envisions sampling of potential target sites by repeated cycles of dissociation/reassociation of proteins with DNA. This mechanism is commonly assumed to be the search strategy used by site-specific DNA-binding proteins. The theoretical underpinnings of this model were developed in the 1970s (see Ref. 1 for review), describing the situation where the protein binds nonspecifically to the DNA, dissociates at some intrinsic rate, and then diffuses through the aqueous medium to sample a second site. This cycle is repeated until a high affinity target site is found. This model predicts that the rate of search will be controlled solely by the intrinsic rate of protein dissociation from nonspecific DNA sites and the speed of diffusion through the nuclear volume.
The need for alternative models was made apparent by the early observations of Riggs et al. (2), indicating that the association rate for Escherichia coli lac repressor binding to its regulatory element was several orders of magnitude faster than that allowed by a simple diffusion-controlled mechanism (3, 4). Further experiments with lac repressor-operator (5, 6) and E. coli CAP protein (7) confirmed and extended this observation.
To account for this discrepancy, a second model for protein-DNA search was devised. This strategy describes an intersegment transfer mechanism requiring two binding sites on the protein (or a protein dimer with one site on each monomer). One of the binding sites releases while the other remains bound, allowing the free binding site to interact with a second strand of DNA. This model, termed intersegment transfer (4, 8), predicts that increasing concentrations of DNA will increase the apparent dissociation rate of the protein; this is in contrast to the dissociation/reassociation model, in which the rate of protein dissociation is intrinsic and not affected by exogenous DNA.
The third mechanism involves the protein sliding along the DNA chain until it encounters a specific binding site. This model reduces the search space to a one-dimensional random walk and has the advantage that it is highly efficient for locating a binding site over a limited distance along the DNA molecule. This model has been successfully applied to restriction endonucleases (9, 10) and the lac operator (11). This mechanism and the first two are not mutually exclusive. Indeed, it is likely that most proteins conduct a short range one-dimensional search in combination with one of the other search mechanisms. Theoretical work indicates that this mechanism alone is not an efficient strategy for a long distance search (12).
Our studies do not directly address the sliding model; instead, they
seek to distinguish whether the intersegment transfer or the cycling
search mechanism may be employed by the steroid receptor family of
transcription factors. We have chosen to study the well characterized
rat glucocorticoid receptor DNA binding domain protein fragment
(GRdbd).1 This fragment, which contains
amino acids 440-525 of the rat GR, contains two zinc finger motifs and
has strong DNA sequence binding specificity (13). The solution
structure of the GRdbd has been determined by NMR, and the crystal
structure of the GRdbd bound to DNA has been deduced (14, 15, 16, 17, 18, 19, 20). The
GRdbd is composed of two zinc finger-stabilized -helices. The first
helix is responsible for specific DNA contacts (14), whereas the second
is positioned away from the DNA at right angles to the first helix and
making no DNA contacts at a target site. The GRdbd is a monomer in
solution and has been shown to bind to DNA both as a monomer and
cooperatively as a dimer (15, 21).
This paper addresses not only the question of whether GRdbd, as a model of the steroid receptor family of transcription factors, may employ an intersegment transfer mechanism as part of its target site search strategy but the mechanistic details of this transfer as well. These questions touch directly upon the ability of regulatory proteins to mediate their effects by rapidly locating specific binding sites in the genome.
Bacterially expressed, purified GRdbd (an 88-residue peptide encompassing the rat GR DNA binding domain from amino acid 440 to amino acid 525) was generously provided by Dr. Len Freedman, Cell Biology and Genetics Program, Sloan-Kettering Cancer Institute, New York, NY. Purified oligonucleotide DNA was purchased from the University of Colorado Cancer Center, Denver, CO, and DNA Express, Colorado State University, Fort Collins, CO. All other reagents were obtained from Sigma, Fisher, and Boehringer-Mannheim unless otherwise noted.
DNA mimics (from Sigma) were heparin (H3125), heparin disaccharide (H9267), poly-L-proline (P2129), poly-L-glutamate (P4886), and 1:1 poly-L-glutamate/glutamate-O-ethyl (P4785).
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The conditions described by Alroy
and Freedman (13) were adapted for these studies. Briefly,
complementary strands of oligonucleotide DNA were allowed to slowly
anneal for 2 h in TEK buffer from 90 °C to room temperature.
Annealed oligonucleotides were labeled with 30 µCi of
[-32P]dCTP (3000 Ci/mmol) and Klenow enzyme. Free
counts were removed with a Sephadex G-25 spin column, followed by
ethanol precipitation in 2.5 M ammonium acetate. The
radioactive pellet was washed once with 70% ethanol, dried, and
resuspended to 5 ng/µl.
For a standard 20-µl binding experiment, purified GRdbd (0.89 ng) and labeled DNA (2.0 ng) were incubated at a 1:1 molar ratio (4.5 nM each) unless otherwise noted, for 30 min at room temperature in GRdbd binding buffer (20 mM Tris-HCl, pH 7.9, 1 mM EDTA, 0.05% Nonidet P-40, 10% glycerol, 50 mM KCl, and 1 mM DTT). Dissociation experiments were conducted by dilution of GRdbd/DNA 100-fold in binding buffer containing 10-fold molar excess of the full binding site oligonucleotide to prevent reassociation of the GRdbd. Protein-DNA complexes were resolved on a precooled 10% nondenaturing polyacrylamide gel (75:1 acryl:bis) and run at 400 V for 2.5 h in 0.5 × TBE buffer (40 mM Tris base, 1 mM EDTA, pH 8.0, 40 mM borate) at 0-4 °C. There was a delay time of ~15 s between dilution of complexes and the time the complexes entered the gel for the nominal zero time point. Gels were fixed for 10 min in 7% acetic acid followed by 10 min in 100% methanol (to prevent cracking). Dried gels were exposed to a phosphorimager screen for 2-18 h, and bound and free DNA were quantified on a Molecular Dynamics PhosphorImager, model 300E (Sunnyvale, CA).
Sheared E. coli DNAA 50 ml overnight bacterial culture
(DH5) in nutrient broth (22) was centrifuged at 3300 rpm (2000 × g) for 10 min to form a pellet. Cells were resuspended in
13.3 ml of TE. To this was added 10% SDS to a final concentration of
0.5%, proteinase K to 200 µg/ml, and RNase to 8 µg/ml. The cells
were incubated at 37 °C for 1 h, and then NaCl was added to 0.7 M and polyethylene glycol 8000 to 0.5% final
concentration; the solution was then shaken at 65 °C for 10 min. The
mixture was then left at room temperature for 30 min. The resulting DNA
was extracted once with an equal volume of chloroform and three times
with phenol/chloroform (1:1) and precipitated with 2 volumes of EtOH at
20 °C overnight. The DNA pellet was centrifuged for 15 min at
14,000 × g, washed with 70% ethanol, dried, and
resuspended in TE. The A260 was determined, and
the DNA was diluted to 5 mg/ml. The DNA was sheared by 20 passages
through a 25 gauge needle; it ranged in size from 500 to 20,000 bp as
determined by gel electrophoresis on a 0.8% agarose gel.
The GRdbd has been shown to exist as a monomer in
solution, but it can bind to DNA cooperatively as a dimer (13, 23, 24, 25, 26, 27, 28).
This is presented schematically in Fig. 1. An
intersegment transfer mechanism can be distinguished from cycling via
repeated dissociation and reassociation with DNA by assessing whether
the dissociation of the bound GRdbd from DNA is influenced by the
concentration of DNA in solution. An intersegment transfer mechanism
predicts that increasing DNA will increase the rate of dissociation,
whereas increasing DNA will have no effect if a cycling mechanism is
used. Experimentally, we measure the loss of the receptor-labeled DNA
complex (R2D) with time at different
concentrations of unlabeled DNA. First, GRdbd is bound to labeled DNA
to equilibrium, and then disequilibrium conditions are imposed so that
reestablishment of R2D* is negligible
once dissociation has occurred. Generally, this is accomplished by blocking reassociation with a large molar excess of unlabeled DNA.
However, this strategy is invalid if DNA itself influences the apparent
dissociation constant. We therefore have determined the association and
dissociation constants for monomer (k1,
k1) and dimer (k2,
k
2) and established conditions wherein we can
measure the apparent dissociation constant in the presence of different
concentrations of DNA.
In Fig. 2 are presented equilibrium binding experiments
at GRdbd concentrations ranging from 5 to 250 nM (1-50 ng)
and oligonucleotides containing either a full glucocorticoid response
element site (right) or a half-site (left). The
gel mobility shift assay was used to separate bound complex from
free DNA, and each was quantified by phosphorimager analysis. A
half-site oligonucleotide was used to measure the equilibrium binding
constant K1 for GRdbd monomer binding to DNA. We
were able to measure nearly pure monomer complex at concentrations of
GRdbd up to 50 nM (10 ng/20 µl). Using the experimentally
determined values of free DNA and GRdbd/DNA complex, the equilibrium
binding constant K1 = [RD]/[D][R] = 1.8 × 107 M1 was derived.
The dimer equilibrium constant K2 was then
determined by measuring the overall equilibrium constant from
experiments in which GRdbd was bound to a full, palindromic binding
site and solving Koverall = K1K2 = [R2D]/[D][R]2
for K2. A value of 3 × 108
M1 was determined for the equilibrium dimer
binding constant K2. The observation that
K2 is greater than K1 is
indicative that dimer binding is cooperative. Data presented in Fig. 2
were used to determine a Hill coefficient (Fig. 3). A
coefficient of 1.5 was determined for the binding of GRdbd to the
full-site oligonucleotide. A Hill coefficient for dimer binding of
between 1 and 2 is indicative of positive cooperativity (29).
To study dissociation of a binding protein from DNA, reassociation must be prevented. This is often accomplished by adding excess, unlabeled competitor DNA to inhibit reassociation with labeled DNA. However, this will give incorrect estimates of dissociation rates if the added DNA itself influences the apparent rate of dissociation, as would be predicted for an intersegment transfer mechanism. Gel shift experiments are often done in conditions of protein excess to maximize signal. However, this also means that high excesses of unlabeled DNA must be used to soak up the excess binding protein, thereby exacerbating the possibility that the apparent dissociation rates are being influenced by the added DNA. An alternative strategy to prevent reassociation is to greatly reduce the concentration of the bound complex by dilution, shifting the equilibrium to favor dissociation. Experimentally, this is limited by the ability to detect binding in extremely dilute solutions.
We have established that we can readily assess GRdbd binding following
a 100-fold dilution of our standard binding condition. To test the
extent to which reassociation occurs at this dilution, we mixed DNA and
GRdbd at concentrations equivalent to the 100-fold diluted standard
condition and showed that some binding was still detectable (about 15%
of that seen with undiluted extract). Therefore, some reassociation can
occur even after dilution. To prevent this small amount of
reassociation, we examined adding a small amount of specific
oligonucleotide to the dilution buffer. In a binding reaction in which
GRdbd is bound to labeled DNA at a 1:1 molar ratio, addition of an
equimolar amount of unlabeled specific oligonucleotide reduces binding
by precisely one-half and a 10-fold excess reduces binding by 90%, a
concentration at which nonspecific oligonucleotide does not compete
(Fig. 4A). In Fig. 4B we show that
addition of a 10-fold molar excess of specific oligonucleotide to the
dilution buffer completely prevents any reassociation. As subsequent
experiments will confirm, addition of this concentration of specific
oligonucleotide is still 200-fold below levels at which effects on
dissociation rates are observed. Therefore, in all subsequent
experiments, dissociation is initiated by 100-fold dilution into a
10-fold excess of specific oligonucleotide. Other nucleic acids or test compounds were added as indicated.
Dissociation Kinetics of GRdbd/DNA Complex
Using the dilution
condition established above, we examined the dissociation of monomer
and dimer GRdbd. As can be seen in Fig. 5, dissociation
of monomer from DNA, unlike that of dimer, is quite rapid. Dissociation
constants were calculated according to the integrated first-order
kinetic equation kdt = ln(Rt/Ro), where
kd is the dissociation constant, t is
time, Rt is the complex remaining at time t, and R0 is the initial bound GRdbd
(taken as bound counts at time 0). In all of the succeeding figures,
dissociation data is presented as ln(Rt)
versus time, and the absolute value of the slope of the best
fit line represents the apparent kd. As shown in
Fig. 5, the dissociation rate of monomer GRdbd from the half-site
(k
1) is 1.2 × 10
2
s
1 (~88 s), and dimer GRdbd from the full site
(k
2) is 2.2 × 10
4
s
1 (~77 min). Thus, the half-life of the monomer
complex (ln(2)/k) is about 60 s, and for the dimer
complex the half-life approaches 60 min.
The association rate for monomer (k1) was too
rapid to be measured by gel shift analysis, and because the association
rate for dimer (k2) is dependent on the
formation of monomer (the GRdbd is a monomer in solution), a direct
measurement of association was not possible. However, using the
relation between the equilibrium constant and the kinetic constants
(K1 = k1/k1, and similarly, K2 = k2/k
2) we calculate the
values for k1 and k2 as
2.1 × 105 and 5.6 × 104
M
1s
1 for monomer and dimer,
respectively. The equilibrium constant for dimer binding is ~17 times
greater than that for monomer binding, indicative of strongly
cooperative binding. This is despite the fact that association of
monomer is 3-4-fold faster than that of dimer. Thus, the cooperative
effect can be attributed to stabilization of the DNA-bound complex upon
dimer formation that results in an almost 60-fold reduction in off-rate
compared to monomer.
Having
established the kinetics of this system, we then measured the effect of
additional DNA on the dissociation rate of dimer. Because the
intersegment transfer mechanism predicts that increasing concentrations
of DNA should increase the apparent dissociation rate of dimer (in
contrast to the cycling model), we tested the ability of sheared
E. coli DNA and specific or nonspecific oligonucleotides to
increase the apparent dissociation rate of GRdbd bound to a specific
oligonucleotide. As seen in Fig. 6, increasing amounts
of sheared E. coli DNA in the dissociation dilution mix
increased the measured off-rate from 2.7 × 104 to
3.6 × 10
3 s
1 (from
t1/2 = 42.8 min to t1/2 = 3.2 min) over the concentration range of 0-3.0 × 10
4
Mbp (0-200 µg/ml). This is a 13-fold increase in the apparent dissociation rate of GRdbd dimer. The same effect can be demonstrated for specific and nonspecific oligonucleotides (data not shown). Increasing concentrations of oligonucleotide DNA from 1.0 × 10
7 to 1.0 × 10
5 M
increases the off-rate up to 5.8 × 10
3
s
1 (t1/2 = 2.0 min) for specific
oligonucleotide, and 6.0 × 10
3 s
1
(t1/2 = 1.9 min) for nonspecific oligonucleotide.
Note that the size of the oligonucleotide used here is 34 base pairs,
so the final concentration of added oligonucleotide DNA falls between 3.4 × 10
6 and 3.4 × 10
4 Mbp
(between 2.2 and 220 µg/ml), an effective range similar to that
obtained with sheared E. coli DNA. These data suggest that the bound GRdbd dimer can transfer between two separate DNA molecules. In addition, the similarity between specific and nonspecific
oligonucleotide results suggests that intersegment transfer occurs via
non-site-specific binding of DNA. This observation has implications for
the binding mechanism employed, an issue addressed further below and
under "Discussion."
GRdbd Monomers Are Capable of Intersegment Transfer
Although our initial expectation was that intersegment transfer would occur by a strand invasion mechanism whereby one monomer of a bound dimer would transiently bind two separate molecules of DNA, the failure of specific oligonucleotide to increase the apparent off-rate any differently than nonspecific oligonucleotide or bulk DNA led us to question whether a domain other than the site-specific DNA binding domain was mediating intersegment transfer and therefore whether a monomer was itself capable of intersegment transfer.
To test this hypothesis, the half-site oligonucleotide was used at
concentrations determined to permit exclusively monomer formation (see
Fig. 2). We observe indications that monomer complex can undergo
intersegment transfer even at added DNA concentrations at the low end
of those at which dimer dissociation was affected. As shown in Fig.
7, nonspecific DNA increased the monomer dissociation rate to the limit of our systems measurement (t1/2 20 s). At DNA concentrations greater than 1 × 10
6 Mbp (22 µg/ml), dissociation was faster than could
be measured. Similar results were obtained with specific
oligonucleotide. These data suggest that monomers as well as dimers of
GRdbd can transfer from one DNA strand to another.
Comparison of Various DNA-like Molecules on Dimer Dissociation Kinetics
To test the specificity of the transfer mechanism, we
employed several compounds that would mimic the long chain polyanionic character of DNA and several control compounds as well (Fig.
8). Three compounds with polyanionic character, heparin,
polyglutamate, and ribo-poly(A)·poly(U) could promote dissociation of
GRdbd from DNA. However, dissociation was not affected by a neutral
macromolecule, polyproline, nor the polyanion subunits, glutamate and
heparin disaccharide. Two cations, arginine and spermidine, likewise
had no effect on dissociation. These data indicate that the interaction of GRdbd that permits intersegment transfer is primarily electrostatic, requiring a long chain polyanion to promote the dissociation of GRdbd
from DNA.
A counterion condensation layer is present around DNA and other long
chain polyions of sufficient charge density. Displacement of this
charge condensation layer provides an entropic gain for protein binding
to DNA. Because the intersegment transfer reaction appears to be via
nonspecific interactions with negatively charged polyanions, our
results suggest that the counter-ion condensation layer around the DNA
may play a role in direct intersegment transfer (see "Discussion").
We wished to test whether there was a density of negative charge
necessary to promote the intersegment transfer mechanism. Using
poly-Glu and a 1:1 random copolymer of Glu/Glu-OEt, we examined the
ability of each compound to mediate intersegment transfer. The results
shown in Fig. 9 indicate that the transfer reaction is
severely inhibited by the reduction of the average charge of the
glutamate backbone by 1/2. Poly-Glu increased the off-rate over
80-fold at the highest concentration tested, whereas the Glu-Glu-OEt
copolymer increased it only 3-fold. These data indicate the importance
of the density of negatively charged residues along the chain, and
implicate the need for the formation of a counter-ion condensation
layer. The residual effect of the poly-Glu/Glu-OEt may be attributable
to the fact that because it is a random copolymer, there will be an
occasional region of high charge density due to the clustering of Glu
residues in the polymer. Nevertheless, it is clear that charge density
is important for the transfer of GRdbd bound to DNA.
The present study confirms earlier work indicating that GRdbd exhibits positive cooperativity in binding to DNA as a dimer. Härd et al. (23) estimated by fluorescent spectroscopy that the cooperative effect was about 10-fold for a somewhat larger GRdbd fragment (115 versus 85 amino acids) binding at high salt conditions (270 mM). More recently, this group updated its findings at lower salt conditions (187 mM), estimating a 25-50-fold cooperativity (17, 18). In our work, the equilibrium constant for dimer binding K2 exceeded that of monomer binding K1 by 15-fold. Moreover, the kinetic work presented here indicates that the cooperative effect is driven by stabilization of the bound dimer (t1/2 = 60 min for dimer dissociation versus t1/2 = 60 s for monomer dissociation).
Search Mechanisms in ChromatinSince the initial observations
by Riggs et al. (2) in the early 1970s that E. coli lac repressor associated with DNA 100-fold faster than
simple diffusion would allow (2), a great deal of theoretical and
experimental work has been conducted on search mechanisms for binding
sites in chromatin. The theoretical framework for diffusion-controlled
association of protein with DNA has been detailed extensively (31, 32, 33, 34).
Based on von Smoluchowski diffusion theory (35), these authors describe the macromolecular and micromolecular interactions that occur when
DNA-binding proteins interact with the DNA chain. Briefly, as a
DNA-binding protein diffuses through a solution, it undergoes collisions with the DNA chain where nonspecific electrostatic contacts
are made (macromolecular collisions). Electrostatic stabilization (with
counter-ion release) can then allow the protein to be localized near
the DNA chain; this results in multiple short range contacts (micromolecular collisions). Thus, the protein can sample several binding orientations before diffusing back into the solution. If the
protein has localized near a binding site, then it can bind this site
with high affinity. If, however, there is no specific site, and no
other mechanisms are proposed to allow protein movement, then the only
method available for further search is to dissociate back into the
medium and rebind at another location. Clearly, this mechanism cannot
account for a number of instances in which association rates of
DNA-binding proteins have been measured that are substantially faster
than diffusion-controlled mechanisms allow (2, 3).
Because the protein initially binds DNA electrostatically, a sliding
model was proposed to facilitate local searches (12, 36). This model
was based on the assumption that nonspecifically bound protein is able
to move freely along the DNA chain by displacing salt ions from in
front and filling the gap behind with no change in the total number of
displaced counter-ions (5). This mechanism has been shown
experimentally with restriction endonucleases (9, 10) and lac
repressor-operator (6, 11). In this fashion, a protein conducts a rapid
(on the order of 109 cm2s
1
(12)) one-dimensional search of the DNA. However, this search only
occurs over a relatively short distance along the DNA strand before the
protein dissociates.
In addition to simple diffusion and sliding, a third mechanism, intersegment transfer, was proposed to facilitate the location of specific sites in the genome. This mechanism envisions that a protein bound to one DNA site transfers directly to a second site via an intermediate stage in which it is transiently bound to two molecules of DNA (5, 8, 34, 37). This strategy reduces the dimensions of the search space and allows the protein to conduct a rapid search for a binding site by maintaining a close association with the DNA matrix. This mechanism also has the advantage that increasing DNA concentration increases the rate of search by enhancing the apparent off-rate of the protein, thus allowing a more rapid sampling of potential binding sites. Solutions to the kinetics equations for a model protein suggest that the rate of site sampling can be increased by 3 orders of magnitude.2
GRdbd Employs Intersegment Transfer as a Search MechanismGlucocorticoid hormone must initiate a series of steps to convert its receptor to an active transcription factor, including dissociation of the receptor from heat shock proteins, translocation of the receptor to the nucleus, location of binding sites on a target promoter, and assembly of a transcription complex. Glucocorticoids can modify rates of transcription within minutes (38), necessitating that the receptor rapidly locate and bind target sites. Our data are not consistent with the concept that the receptor finds these sites by sampling through repeated rounds of association and dissociation. The intrinsic dissociation rate of GRdbd dimer from DNA is slow; however, the apparent dissociation rate is greatly enhanced by DNA, a result consistent with our interpretation that the GRdbd is employing intersegment transfer as a search mechanism. This enhancement was detected at DNA concentrations ranging from 2 to 200 µg/ml. The concentration of DNA in the nucleus of a eukaryotic cell is estimated at ~10-50 mg/ml. This presents a physical obstacle to diffusion-controlled search strategies and an aid to an intersegment transfer mechanism. Even acknowledging that a fraction of DNA may be packaged so that it is effectively inaccessible to receptor, intersegment transfer is a particularly effective mechanism for exploiting the circumstances posed by the eukaryotic nucleus in undertaking a search for a target site.
Analysis of the crystal structure of the GRdbd·DNA complex indicates
that specific DNA contacts are made via an -helical domain present
at the C-terminal end of the first zinc coordination finger (14). The
intersegment transfer mechanism requires that GRdbd transiently bind
two separate molecules of DNA. It is a straightforward assumption that
one monomer of a DNA-bound GRdbd dimer could release from its site and
bind a second DNA followed by transfer of the other half of the dimer
to the second DNA. However, this supposition is inconsistent with the
finding that the dissociation from a specific target is enhanced
equally by a nonspecific oligonucleotide and by one containing a
specific target site. If transfer involves the DNA binding domain that mediates specific binding, one would anticipate that transfer would be
less favored if specific contacts could not be made. Indeed, transfer
can be mediated by a variety of polyanions. Moreover, our data indicate
that the dissociation of monomer from DNA is enhanced by DNA,
suggesting that the monomer itself can transiently bind separate
molecules of DNA. Therefore, the GRdbd monomer must posses a second
domain capable of nonspecific interaction with DNA. There are two
possibilities for candidates for this domain. The crystallography data
(14) indicate that the
-helix at the C-terminal end of the second
zinc finger makes no DNA contacts and that when the GRdbd is bound to a
specific site, the second helix is positioned away from and at right
angles to the first helix. A peptide comprising the second zinc finger
can bind DNA (39), and helix-disrupting mutations in this helix
abrogate function in the context of the intact receptor (40). Thus,
this helix is potentially responsible for mediating transfer through nonspecific DNA binding. A second domain that could assist with or
mediate nonspecific DNA binding is a highly basic region near the C
terminus of the GRdbd that was not localized in the crystal structure.
In preliminary studies, a GRdbd fragment lacking this region (courtesy
of T. Kerppola) failed to bind DNA.
During a search for target sites in vivo, a glucocorticoid receptor would first sample and transfer from many nonspecific sites before locating a specific target site. Binding of GRdbd to nonspecific DNA was too low affinity to permit its quantification and therefore assess whether DNA could influence the apparent off-rate. Therefore, the experiments above were conducted by initially binding GRdbd to a palindromic target site. Thus, it is clear that DNA at moderate to high concentrations enhances the apparent off-rate of GRdbd from specific target sites, as well as from nonspecific sites. It is possible that assembly of the receptor into a transcription complex at its target site abrogates intersegment transfer, perhaps because the second DNA binding domain necessary for the transient, simultaneous binding to two DNA molecules is involved in protein-protein interactions in such a complex. Alternatively, cycling on and off a target site may be critical to receptor action. One can envision mechanisms whereby the receptor acts as a single-shot effector that must be removed and reloaded onto a target site before it is capable of again fulfilling its activation function. In this case, intersegment transfer may be critical not just to the search for target sites but to the transcription activation function of the receptor at the target site as well.
The DNA mimic experiments (Figs. 8 and 9) indicate that there is
surprisingly little constraint on the structure of the molecule that
will promote dissociation of GRdbd from DNA. RNA, heparin, and
polyglutamate all increase the apparent off-rate. This result supports
the hypothesis that the second interaction is primarily electrostatic,
requiring a long chain polyanion. Decreasing charge density inhibits
the ability of the polyanion to mediate the transfer (Fig. 9). Based on
theoretical analyses (41, 42), the amount of thermodynamically bound
counterions around a long chain molecule (e.g. DNA) is
dependent on the average charge separation along the chain. Release of
counter-ions from the DNA by protein binding has been shown to create
an entropic stabilizing force for nonspecific protein-DNA interactions
(41, 42). For double-helical B-DNA this charge separation is 1.7 Å (43), and results in a fractional charge neutralization () of 0.88. The structure of poly-Glu is not known, but if we assume an extended
chain, then the average charge separation can be represented by the
-carbon of the side chain projected back to a central line along the
polypeptide backbone, and the charge separation should be about 3.62 Å (30). This gives a linear charge density of
~ 1.96 (43), and the
fractional charge neutralization (
) is 1
(1/1.96) = 0.49, indicating that poly-Glu has the capacity to form a thermodynamically
bound ion condensation layer. The substituted polypeptide (1:1)
poly-Glu/Glu-OEt, in which half of the charged carboxyl groups have
been replaced with an ester linked ethyl group, has an average charge
separation of ~7.2 Å and thus should have no counter-ion
condensation layer associated (
~ 1.0;
= 0) (43). This random
copolymer retains some small ability to mediate transfer, probably
through scattered regions of clustered Glu residues, where the local
charge density is high enough to create regions of organized
counterions.
Dissociation
measurements with the GRdbd also point out a potential problem
encountered by experiments designed to measure dissociation rates of
DNA-binding proteins. Many researchers use a large excess of specific
competitor DNA in their experiments to prevent reassociation of the
protein with a labeled specific DNA fragment. Because we have shown
that the concentration of competitor DNA added to a protein-DNA binding
reaction can potentially affect the rate of apparent dissociation,
experiments using excess competitor DNA to measure dissociation must
experimentally determine the minimal amount of necessary competitor in
order to avoid a systematic error. In our experiments, with the GRdbd
at 1:1 molar ratio to DNA, we determined that 2-fold excess specific
competitor was sufficient to disrupt the binding equilibrium, and
10-fold excess specific competitor was sufficient to prevent
reassociation to labeled DNA (Fig. 4). This latter concentration of
specific competitor DNA (50 nM for standard conditions and
1/100 of that for the diluted dissociation conditions) is below that
seen to influence the off-rate by intersegment transfer
(>107 M or 2-3 × 10
6
Mbp). For example, if a typical experiment using 1 ng of bound 30-mer
oligonucleotide was competed with 400-fold competitor, then a 20-µl
sample volume would contain 10
6 M
oligonucleotide (3 × 10
5 Mbp), a concentration
sufficient to substantially influence the dissociation rate of GRdbd.
If the binding reaction contained nonspecific competitor, such as
poly(dI·dC), which is often used to reduce the level of nonspecific
protein binding from crude protein preparations, then this also could
influence the measured equilibrium and dissociation kinetics.
We have presented data that are compatible with the theory that the GRdbd is able to transfer directly from one DNA molecule to another using an intersegment transfer mechanism. We propose that the active full-length GR is able to sample potential target sites rapidly by a similar mechanism. At a target promoter, the association of GR with transcriptional coactivators may stabilize GR dissociation from the site by abrogating intersegment transfer, perhaps because the protein-protein interactions sterically hinder the nonspecific DNA binding site needed to conduct intersegment transfer. Alternatively, receptor could activate transcription by a single-shot mechanism so that rapid transfer from DNA would be required to remove receptor from a target site in order for it to be reloaded and again fired. Although the intersegment transfer mechanism must still be shown with the full-length receptor, we are attracted to the idea of the receptor swinging through the nuclear jungle from DNA to DNA in search of a specific binding site.
The authors gratefully thank our colleagues Dr. David Pettijohn for introducing us to the concept of intersegment transfer, Dr. John Cann for sharing his insight in enzyme kinetics, and Dr. Len Freedman for providing the purified GRdbd protein fragment for these studies.