(Received for publication, January 23, 1997, and in revised form, May 22, 1997)
From the Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461
The rates of association of the tetrameric Lac repressor (LacI), dimeric LacIadi (a deletion mutant of LacI), and the native dimeric Gal repressor (GalR) to DNA restriction fragments containing a single specific site were investigated using a quench-flow DNase I "footprinting" technique. The dimeric proteins, LacIadi and GalR, and tetrameric LacI possess one and two DNA binding sites, respectively. The nanomolar protein concentrations used in these studies ensured that the state of oligomerization of each protein was predominantly either dimeric or tetrameric, respectively. The bimolecular association rate constants (ka) determined for the LacI tetramer exceed those of the dimeric proteins. The values of ka obtained for LacI, LacIadi, and GalR display different dependences on [KCl]. For LacIadi and GalR, they diminish as [KCl] increases from 25 mM to 200 mM, approaching rates predicted for three-dimensional diffusion. In contrast, the ka values determined for the tetrameric LacI remain constant up to 300 mM [KCl], the highest salt concentration that could be investigated by quench-flow footprinting. The enhanced rate of association of the tetramer relative to the dimeric proteins can be modeled by enhanced "sliding" (Berg, O. G., Winter, R. B., and von Hippel, P. H. (1981) Biochemistry 20, 6929-6948) of the LacI tetramer relative to the LacIadi dimer or a combination of enhanced sliding and the superimposition of "direct transfer" mediated by the bidentate DNA interactions of the tetramer.
It is well established that the binding of a protein to a specific sequence of DNA can, under some experimental conditions, proceed at rates significantly faster than those predicted by three-dimensional diffusion. This phenomenon is referred to as "facilitated diffusion". A well studied example is the association of the Escherichia coli Lac repressor (LacI)1 with its operators (1-4). Models have been proposed and tested whereby an initial nonspecific binding to a DNA molecule is followed by one or more mechanisms by which a protein translocates along the DNA molecule to the specific binding sequence (2, 5-9).
The term "sliding" describes the one-dimensional diffusion of nonspecifically bound proteins along DNA. Characteristics of this mechanism are its sensitivity to monovalent ion concentration and its dependence on the length of the DNA molecule. Its generality is indicated by results obtained for Cro repressor (10), the restriction enzyme EcoRI (11, 12), and E. coli RNA polymerase (13). "Direct transfer" is a mechanism of facilitated diffusion whereby a DNA-bound ligand or protein transiently binds to two DNA segments simultaneously (14, 15). The binding of the bidentate protein, such as LacI, to two sites on a single DNA molecule can result in the formation of a DNA loop, allowing the protein to sample distant DNA sequences simultaneously (16-18).
Investigations into the relative contributions of the sliding and direct transfer mechanisms using LacI and a dimeric mutant protein, LacIadi, have been conducted (19, 20). However, linked self-association reactions may have complicated these comparisons. To circumvent this issue, a quench-flow DNase I "footprinting" technique (21) has been utilized to conduct kinetic studies of LacI and LacIadi at concentrations sufficient to ensure that the proteins were predominantly tetrameric and dimeric, respectively. The E. coli Gal repressor (GalR) was used as an additional model of a protein whose native form is a monodentate dimer (22). Primary sequence alignments, molecular modeling, and x-ray diffraction studies strongly suggest that GalR and LacI share similar tertiary structures (22-26).
These studies demonstrate that the LacI tetramer binds operator more rapidly than the LacIadi dimer. Simulations conduct using the model of Berg et al. (2) suggest that direct transfer alone cannot account for the salt dependence of the LacI tetramer rate enhancement. Rather, an increase in the sliding rate for the LacI tetramer relative to the LacIadi and GalR dimers is required to account fully for the differences in association rates of the proteins.
The GalR used in these studies was prepared following published protocols (22). The LacIadi preparation was a gift from Dr. Sankar Adhya. The LacI used in these studies was obtained by expression in E. coli of plasmid pMB12 and purified as described elsewhere (27). The sequence-specific DNA binding activities of the proteins were determined from binding experiments conducted under conditions where the DNA concentration exceeds the equilibrium dissociation constant.
DNAThe 32P-labeled DNA restriction fragments
used in these studies (28, 29) were present at concentrations of 10-20
pM in the reaction mixture. DNA restriction fragments
of three lengths (185, 635, and 2,900 bp) containing the gal
promoter region were excised from the parent plasmids (Fig.
1 and Refs. 29 and 30). For the GalR
binding kinetics experiments, the restriction fragments excised from
plasmid pDW001 contain the native gal operator at OE (OEG;
GTGTAAAC|GATTCCAC). The sequence of the second operator,
OI, whose center of symmetry is located 11 helical turns downstream from that of OE,
is not capable of binding GalR by virtue of its having been replaced
with a sequence recognized with high affinity by LacI at
OI (OIL;
TTGTGAGC|GCTCACAA). For the LacI and LacIadi
binding kinetics experiments, restriction fragments from plasmid pDW002
have the LacI recognition sequence substituted at
OE (OEL;
TTGTGAGC|GCTCACAA) and the native GalR sequence at
OI. Thus, each restriction fragment contains a
single specific site for the protein being analyzed.
Kinetic DNase I Footprinting
Kinetics progress curves were obtained by quantitative DNase I footprinting implemented using a quench-flow protocol that is described in detail elsewhere (21). Briefly, a three-syringe quench-flow apparatus (Kin-Tek®) is used. Two drive syringes containing assay buffer (25 mM bis-tris, 5 mM MgCl2, 2 mM CaCl2, 1 mM dithiothreitol, pH 7.0, at 20 °C with KCl concentrations from 25 mM to 300 mM) propel the DNA and protein samples contained in the "sample loops" into a "reaction loop" where they combine. After a programmed pause interval, the protein-DNA mixture is propelled into contact with the DNase I solution, supplied by the third drive syringe, at the end of the reaction loop. The third syringe is filled with a concentration of DNase I sufficient for a "single hit" DNA cleavage reaction as the samples and DNase I mixture move through the exit line. The DNase I cleavage reaction is quenched when the reaction mixture is expelled from the exit line into an EDTA-containing solution present in the microcentrifuge collection tube. The microcentrifuge tube is placed in a dry ice-ethanol bath, and the 32P-DNA reaction products are processed for analysis by denaturing gel electrophoresis (21, 28).
Densitometry and Progress Curve AnalysisDigital images of
the electrophoresis gels was obtained by Phosphor storage
autoradiography using a Molecular Dynamics PhosphorImager. Quantitative
densitometry was conducted using the ImageQuant (Molecular Dynamics)
software following published protocols (31-33). The pseudo-first order
approximation was used in the analysis of the kinetics progress curves
since [[32P]DNA] [protein]. The contribution of
the reverse reaction was neglected in the determination of the
association rate constant because kd
ka (1, 28, 34). Densitometric
analysis of the digital images of the footprint electrophoretograms
yields pi, the "apparent
saturation."
, the fractional saturation of
the binding site, is obtained by fitting the data to the coupled equations,
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(Eq. 1) |
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(Eq. 2) |
The kinetics of binding the LacI tetramer and the
LacIadi and GalR dimers to DNA restriction
fragments containing a single operator site were followed using the
quench-flow footprinting technique. An example of an autoradiogram of a
kinetic footprinting experiment is shown in Fig.
2A. Time-dependent
changes in the protection of bases outside the specific binding sites
of the repressors were not observed in any of the experiments (data not
shown). The increase in site-specific protection with time is
quantitated to produce progress curves for each reaction (Fig.
2B). All of the progress curves determined in these studies
are adequately described by a single exponential function; no evidence
of additional kinetic phases was present under any of the experimental
conditions (Fig. 2B).
Extensive thermodynamic data exists for the site-specific binding of
these proteins to DNA (3, 28, 36-42). LacI has been shown to form a
bidentate "looped complex," bridging
OEL and
OIL, on DNA restriction fragments of
length identical to those used in the present study which contain two
operators competent to bind LacI (28, 30, 37). Thus, LacI can form
stable bidentate interactions on even the shortest (185 bp) restriction
fragment used in these studies. The values of
ka determined as a function of
[KCl] for the 185-bp restriction fragment are shown in Fig. 3. The values of
ka obtained for LacI exceed those
obtained for LacIadi and GalR over the entire
[KCl] range. The values of ka
determined are significantly faster than that predicted by
three-dimensional diffusion except above 200 mM KCl for
LacIadi and GalR where
ka is reduced to the range of
diffusion-limited reactions (107-108
M1 s
1). In contrast, the values
of ka determined for LacI exhibit no dependence upon [KCl] from 25 to 300 mM KCl within
experimental error. The differences in
ka between the dimeric and tetrameric proteins are also present in binding reactions conducted with 635- and
2,900-bp DNA restriction fragments at 25 and 100 mM KCl (Fig. 4).
The faster-than-diffusion association rates of LacI to operator-containing DNA have long been appreciated (1-9, 34). A two-step model of association has been proposed and tested,
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Another mechanism, direct transfer, is postulated to occur when a bidentate protein molecule, such as LacI, mediates the formation of a transient "DNA loop." Utilization of this kinetic mechanism by LacI has not been demonstrated unequivocally, although stable protein-mediated DNA loops occur with LacI bound to DNA containing two operators (16-18, 28, 37, 45, 46). The ability of excess operator-containing DNA to enhance dissociation by direct competition suggests that the direct transfer reaction might play a role in the binding of LacI to operator (15, 19). Thus, a plausible hypothesis is that dissociation of the LacI tetramer to dimers would eliminate the direct transfer mechanism while not affecting sliding.
Studies of LacIadi were conducted to compare
directly the kinetic properties of this dimeric form of LacI with the
tetramer (19, 20). A possible complication in these studies is the
uncertain oligomeric states of the proteins at the extremely low
protein concentrations necessary for manual mixing protocols. The
pioneering nitrocellulose filter binding studies of the kinetics of
LacI DNA binding (1-4, 34) used LacI concentrations on the order of
1012 M. The more recent comparisons of DNA
binding by LacI and LacIadi utilized protein
concentrations as high as 10
9 M (19, 20),
well below indirect estimates of 10
8-10
12
M for the Kd of the LacI dimer-tetramer
equilibrium (20, 37, 47). However, a recent analysis of dimer-tetramer linkage with sequence-specific DNA binding suggests that LacI dimers
are not present even at concentrations of 10
12
M (48). In addition to eliminating dimer-to-tetramer
association, the deletion of the COOH-terminal residues of LacI which
create the LacIadi mutant also weakens the
monomer-dimer association (38). A Kd of 7.7 × 10
8 M was determined for this equilibrium
(39). Thus, the previous studies (19, 20) may have been influenced by
the coupled monomer-dimer association reaction of this protein.
The use of a quench-flow apparatus allowed experiments to be conducted
at higher LacI and LacIadi concentrations where
their oligomeric states were well defined. For LacI, concentrations in
the range of 6.3-25 × 109 M were shown
to be optimal for the formation of stable LacI-mediated looped
complexes using the DNA restriction fragments and under the
experimental conditions used in these studies (28, 37). LacIadi is dimeric in the concentration range of
25-100 × 10
9 M employed in these
studies. In addition, high protein concentrations were required to
saturate the operator with LacIadi since the
linked monomer-dimer equilibrium results in diminished apparent binding
affinity (38, 39).
The data presented in Figs. 3 and 4 confirm the differences in ka and reveal different salt dependences for the dimeric LacIadi and tetrameric LacI. These data also confirm the faster-than-diffusion-limited rates obtained using other methodologies, although the ka values for LacI determined are slightly lower than those determined using a 203-bp fragment containing a natural lac operator (34). A comparison of rates between the past (34) and present studies must consider the different experimental conditions and methodologies employed to assay binding and the protocols used to produce and purify the LacI protein. Technical considerations prevented obtaining quench-flow footprinting data at either the picomolar protein concentrations used in the filter binding studies or at [KCl] > 300 mM. The latter limitation was the result of the excessive DNase I concentrations required for cleavage at [KCl] > 300 mM on the millisecond time scale (data not shown). Thus, the direct comparison of the dimeric and tetrameric proteins within the present data set is essential to the interpretation of the data.
The association rate constants obtained for tetrameric and dimeric
proteins are both comparable to or greater than a diffusion-limited case, consistent with a facilitating mechanism for both the dimeric and
tetrameric proteins. The values of ka
obtained for the dimeric proteins demonstrate the [KCl] dependence
anticipated for a binding reaction facilitated by sliding, decreasing
to a diffusion-limited level (107-108
M1 s
1; 2, 7) at 200 mM KCl (Fig. 3). The independence of
ka for the tetramer over this [KCl]
range was unexpected. The model of Berg et al. (2)
postulates the observed value of ka
may be dependent upon sliding, direct transfer, or both mechanisms. The
model is dependent upon the physical properties of the DNA double helix and the free diffusion constants for the proteins. The values for these
parameters were taken from Ref. 2 for the simulations described below.
The value for the nonspecific binding constant, KRD, was calculated from Ref. 49. The
expression for KRD is approximate at
low salt concentrations and in the presence of Mg2+.
However, the inclusion of these effects had no effect on the simulations (data not shown).
The goal of these simulations was to determine whether the values of
ka determined for LacI could be
described by superimposing a direct transfer rate constant (,
s
1) upon the linear diffusion rate constant
(D1, cm2 s
1)
determined from LacIadi data. Because of the
high correlation between
and D1, it is not
possible to determine both constants simultaneously. Thus, the
assumption was made that LacIadi binding is
facilitated solely by sliding. In the absence of direct transfer (
= 0), the values of ka determined for
LacIadi as a function of [KCl] and DNA length
are described by values of D1 ranging from
3 × 10
14 to 2 × 10
13
cm2 s
1 (Figs. 3 and 4). This range of values
is less than that minimally required to describe the binding of the
tetramer LacI as described below.
The dotted lines in Fig. 3 represent simulations in which
increasing values of the direct transfer constant ( = 10 and 1,000 transfers s
1, the latter value being theoretical upper
limit for
; 2), are superimposed upon the value of
D1 of 3 × 10
14
cm2 s
1 which describes the [KCl] dependence
of binding of the dimeric LacIadi. This
combination of sliding and direct transfer accounts for the rate
enhancement of LacI relative to LacIadi at low
salt (<75 mM KCl) but not at high salt. The simulated curve approaches an asymptotic limit that is divergent from the LacI
data in the higher salt concentrations. Thus, an increase in
alone
does not account for the relative difference in association rates
observed between LacI and LacIadi (Fig. 3).
Simulations of the LacI data with D1 ranging
from 3 × 1010 to 7 × 10
11
cm2 s
1 and
= 0 (Figs. 3 and 4,
solid lines) depict the increase in sliding in the absence
of direct transfer which can account for the LacI data. Although the
LacI data can also be modeled adequately by combinations of
and
D1, an increase in D1
appears to be a necessary component of LacI rate enhancement relative
to LacIadi. Because of the high theoretical
correlation between
and D1, it is not
possible to determine unique values for the two parameters. Thus,
although the association of the LacI tetramer is enhanced relative to
the LacIadi and GalR dimers, the portion (if
any) of this enhancement caused by direct transfer cannot be determined
from these data.
The simplest conclusion is that sliding by LacI is enhanced relative to
the LacIadi (Fig. 4), although both the direct
transfer and sliding mechanisms may contribute to the rate enhancement
observed for the tetramer. The LacI tetramer does not appear to be
simply a dimer of LacIadi dimers. However, these
differences do not appear to be manifest at the protein-DNA interface.
Thermodynamic studies of the salt dependence of sequence-specific
binding have been conducted for a 18 COOH-terminal deletion of LacI
whose properties are comparable to the LacIadi
protein (41). At [KCl] > 50 mM, similar ion
stoichiometries were observed for the formation of DNA
sequence-specific complexes for the dimeric and tetrameric proteins,
evidence that the protein-DNA contacts are homologous. (It was also
observed in these studies (41), that the net ion stoichiometries of the
two proteins differed at low ion concentrations, conditions under which
cation binding by the protein is significant.)
Structural and thermodynamic studies suggest a diminished role for direct transfer in the rate enhancement of LacI binding. The recently solved co-crystal structure of LacI (50) revealed an unanticipated arrangement of the dimers within the tetramer in which the two DNA strands are adjacent rather than on opposite sides of the protein. This structure requires that a single DNA molecule assume a fairly complex geometry when the protein bridges two binding sites. A model for a LacI-mediated looped complex based upon the co-crystal structure proposes that the DNA is wrapped around the outside of the LacI tetramer (50). Thus, a fairly complex geometry may be required for the formation of bidentate interactions. It is also plausible that protein-DNA contacts made upon the wrapping of the DNA around LacI could facilitate sliding by the tetramer. In addition, thermodynamic studies have demonstrated that the binding of a second operator to the LacI tetramer is highly salt-dependent and strongly anticooperative at low salt (48). If the observed anticooperativity of second operator binding also applies to nonspecific DNA binding, then a contribution to rate enhancement by direct transfer would be expected to be minimal over the range of KCl concentrations studied. In conclusion, the association kinetics of the LacI tetramer and the LacIadi dimer clearly differ. However, this difference cannot be unambiguously ascribed to the direct transfer mechanism and suggests that the interactions between LacI and DNA contributing to operator location may be more complex than originally thought.