Advertisement

Comparison of the DNA Association Kinetics of the Lac Repressor Tetramer, Its Dimeric Mutant LacI adi, and the Native Dimeric Gal Repressor*

  • Mark Hsieh
    Affiliations
    Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461
    Search for articles by this author
  • Michael Brenowitz
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-3179; Fax: 718-892-0703
    Affiliations
    Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461
    Search for articles by this author
  • Author Footnotes
    * These studies were supported by Grants GM39929 and F31-GM13850 from the National Institutes of Health and by the Hirshl Weill-Caulier Trust. Some of the data in this paper are part of a thesis presented by M. H. for the degree of Doctor of Philosophy in the Sue Golding Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:August 29, 1997DOI:https://doi.org/10.1074/jbc.272.35.22092
      The rates of association of the tetrameric Lac repressor (LacI), dimeric LacI adi (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, LacI adi 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 ofka obtained for LacI, LacI adi, and GalR display different dependences on [KCl]. For LacI adi 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 LacI adi 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 theEscherichia coli Lac repressor (LacI)
      The abbreviations used are: LacI, Lac repressor; GalR, Gal repressor; bp, base pair(s); bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.
      1The abbreviations used are: LacI, Lac repressor; GalR, Gal repressor; bp, base pair(s); bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.
      with its operators (
      • Riggs A.D.
      • Bourgeois S.
      • Cohn M.
      ,
      • Berg O.G.
      • Winter R.B.
      • von Hippel P.H.
      ,
      • Winter R.B.
      • von Hippel P.H.
      ,
      • Barkley M.D.
      ). 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 (
      • Berg O.G.
      • Winter R.B.
      • von Hippel P.H.
      ,
      • Berg O.G.
      • Winter R.B.
      • von Hippel P.H.
      ,
      • Berg O.G.
      • von Hippel P.H.
      ,
      • Berg O.G.
      • von Hippel P.H.
      ,
      • von Hippel P.H.
      • Berg O.G.
      ,
      • Mazur S.J.
      • Record Jr., M.T.
      ).
      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 (
      • Kim J.G.
      • Takeda Y.
      • Matthews B.W.
      • Anderson W.F.
      ), the restriction enzyme EcoRI (
      • Jack W.E.
      • Terry B.J.
      • Modrich P.
      ,
      • Terry B.J.
      • Jack W.E.
      • Modrich P.
      ), and E. coli RNA polymerase (
      • Kabata H.
      • Kurosawa O.
      • Arai I.
      • Washizu M.
      • Margarson S.A.
      • Glass R.E.
      • Shimamoto N.
      ). “Direct transfer” is a mechanism of facilitated diffusion whereby a DNA-bound ligand or protein transiently binds to two DNA segments simultaneously (
      • Bresloff J.L.
      • Crothers D.M.
      ,
      • Fried M.G.
      • Crothers D.M.
      ). 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 (
      • Mossing M.C.
      • Record Jr., M.T.
      ,
      • Mossing M.C.
      • Record Jr., M.T.
      ,
      • Whitson P.A.
      • Olson J.S.
      • Matthews K.S.
      ).
      Investigations into the relative contributions of the sliding and direct transfer mechanisms using LacI and a dimeric mutant protein, LacI adi, have been conducted (
      • Ruusala T.
      • Crothers D.M.
      ,
      • Fickert R.
      • Müller-Hill B.
      ). However, linked self-association reactions may have complicated these comparisons. To circumvent this issue, a quench-flow DNase I “footprinting” technique (
      • Hsieh M.
      • Brenowitz M.
      ) has been utilized to conduct kinetic studies of LacI and LacI adi 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 (
      • Hsieh M.
      • Hensley P.
      • Brenowitz M.
      • Fetrow J.S.
      ). Primary sequence alignments, molecular modeling, and x-ray diffraction studies strongly suggest that GalR and LacI share similar tertiary structures (
      • Hsieh M.
      • Hensley P.
      • Brenowitz M.
      • Fetrow J.S.
      ,
      • Weickert M.J.
      • Adhya S.
      ,
      • Nichols J.C.
      • Vyas N.K.
      • Quiocho F.A.
      • Matthews K.S.
      ,
      • Schumacher M.A.
      • Choi K.Y.
      • Zalkin H.
      • Brennan R.G.
      ,
      • Friedman A.M.
      • Fischmann T.O.
      • Steitz T.A.
      ).
      These studies demonstrate that the LacI tetramer binds operator more rapidly than the LacI adi dimer. Simulations conduct using the model of Berg et al. (
      • Berg O.G.
      • Winter R.B.
      • von Hippel P.H.
      ) 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 LacI adiand GalR dimers is required to account fully for the differences in association rates of the proteins.

      RESULTS

      The kinetics of binding the LacI tetramer and the LacI adi 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. 2 A. 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. 2 B). 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. 2 B).
      Figure thumbnail gr2
      Figure 2A, autoradiogram of a quench-flow kinetics footprinting experiment of the association of 6.3 nm LacI to a 185-bp restriction fragment at 50 mm KCl as described under “Experimental Procedures.” The time scale of the experiment is 0–120 s. B, kinetic progress curve determined for the pictured autoradiogram. Thesolid line depicts the best fit to a single exponential function (Equations and ).
      Extensive thermodynamic data exists for the site-specific binding of these proteins to DNA (
      • Winter R.B.
      • von Hippel P.H.
      ,
      • Brenowitz M.
      • Jamison E.
      ,
      • Record M.T.
      • deHaseth P.L.
      • Lohman T.M.
      ,
      • Brenowitz M.
      • Pickar A.
      • Jamison E.
      ,
      • Brenowitz M.
      • Mandal N.
      • Pickar A.
      • Jamison E.
      • Adhya S.
      ,
      • Chen J.
      • Matthews K.S.
      ,
      • Garner M.M.
      • Rau D.C.
      ,
      • Stickle D.F.
      • Liu G.
      • Fried M.G.
      ,
      • Vossen K.M.
      • Stickle D.F.
      • Fried M.G.
      ). LacI has been shown to form a bidentate “looped complex,” bridgingOEL andOIL, on DNA restriction fragments of length identical to those used in the present study which contain two operators competent to bind LacI (
      • Brenowitz M.
      • Jamison E.
      ,
      • Dalma-Weiszhausz D.D.
      • Brenowitz M.
      ,
      • Brenowitz M.
      • Pickar A.
      • Jamison E.
      ). Thus, LacI can form stable bidentate interactions on even the shortest (185 bp) restriction fragment used in these studies. The values ofka determined as a function of [KCl] for the 185-bp restriction fragment are shown in Fig. 3. The values ofka obtained for LacI exceed those obtained for LacI adi 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 LacI adi and GalR whereka is reduced to the range of diffusion-limited reactions (107–108m−1 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 inka 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).
      Figure thumbnail gr3
      Figure 3The second-order association rate constants (ka ) determined a function of KCl concentration for the binding of LacI (•), LacI adi (○), and GalR (▵) to the 185-bp DNA restriction fragment containing a single operator.Each data point represents the results obtained from two or more independent determinations. For the simulations of both LacI and LacI adi, D = 5 × 10−7cm2 s−1, [DNA] = 2 × 10−11m, a = 500 Å (persistence length), b = 15 Å (DNA radius),l = 3.4 Å (base stack height), and M (DNA length) = 185, 635, or 2,900 bp (
      • Berg O.G.
      • Winter R.B.
      • von Hippel P.H.
      ). The lines denote the results of simulations for the model of Berg et al. (
      • Berg O.G.
      • Winter R.B.
      • von Hippel P.H.
      ). The simulation for LacI adi (– – –) was generated using D1 = 3 × 10−14cm2 s−1 and ν = 0. Simulations for LacI were generated using D1 = 3 × 10−14 cm2 s−1 and ν = 10 s−1 (· · ·) or 1,000 s−1(– · · –) and using D1 = 9 × 10−10 cm2 s−1 and ν = 0 (⎻⎻).
      Figure thumbnail gr4
      Figure 4Comparison of ka values obtained for LacI (•, ▪) and LacIadi (○, □) as a function of DNA fragment length at 25 mm (▪, □) and 100 mm KCl (•, ○). The simulated curves were determined (
      • Berg O.G.
      • Winter R.B.
      • von Hippel P.H.
      ) using values of D1 of 7 × 10−11 cm2 s−1 (•), 3 × 10−10 cm2 s−1(▪), 5 × 10−14 cm2 s−1(○), 2 × 10−13 cm2 s−1 (□).

      DISCUSSION

      The faster-than-diffusion association rates of LacI to operator-containing DNA have long been appreciated (
      • Riggs A.D.
      • Bourgeois S.
      • Cohn M.
      ,
      • Berg O.G.
      • Winter R.B.
      • von Hippel P.H.
      ,
      • Winter R.B.
      • von Hippel P.H.
      ,
      • Barkley M.D.
      ,
      • Berg O.G.
      • Winter R.B.
      • von Hippel P.H.
      ,
      • Berg O.G.
      • von Hippel P.H.
      ,
      • Berg O.G.
      • von Hippel P.H.
      ,
      • von Hippel P.H.
      • Berg O.G.
      ,
      • Mazur S.J.
      • Record Jr., M.T.
      ,
      • Winter R.B.
      • Berg O.G.
      • von Hippel P.H.
      ). A two-step model of association has been proposed and tested,
      R+DOk1k1RDOk2k2ROD


      MODEL1


      where R is the repressor, D is nonspecific DNA, and O is the operator (
      • Berg O.G.
      • Winter R.B.
      • von Hippel P.H.
      ,
      • Mazur S.J.
      • Record Jr., M.T.
      ). In this model, the initial step, k1, is the diffusion-limited nonspecific association of the repressor to the DNA molecule. The second step, k2, is the facilitated translocation of the repressor from the nonspecific binding site to the operator. One mechanism of this translocation is sliding in which the repressor is postulated to diffuse along the DNA to the operator. This mechanism has been inferred from observation of salt and DNA fragment length dependences of the rate constants (
      • Riggs A.D.
      • Bourgeois S.
      • Cohn M.
      ,
      • Winter R.B.
      • von Hippel P.H.
      ,
      • Barkley M.D.
      ,
      • Kim J.G.
      • Takeda Y.
      • Matthews B.W.
      • Anderson W.F.
      ,
      • Jack W.E.
      • Terry B.J.
      • Modrich P.
      ,
      • Terry B.J.
      • Jack W.E.
      • Modrich P.
      ,
      • Whitson P.A.
      • Olson J.S.
      • Matthews K.S.
      ,
      • Winter R.B.
      • Berg O.G.
      • von Hippel P.H.
      ,
      • Khoury A.M.
      • Lee H.J.
      • Lillis M.
      • Lu P.
      ). The physical nature of sliding is uncertain. It has been demonstrated that EcoRI translocates along the helical pitch of the DNA (
      • Jeltsch A.
      • Alves J.
      • Wolfes H.
      • Maass G.
      • Pingoud A.
      ). In contrast, linear tracking of RNA polymerase along straight DNA “brushes” has been observed by fluorescence microscopy (
      • Kabata H.
      • Kurosawa O.
      • Arai I.
      • Washizu M.
      • Margarson S.A.
      • Glass R.E.
      • Shimamoto N.
      ), suggesting that the polymerase does not follow the helical contour of the affixed DNA.
      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 (
      • Mossing M.C.
      • Record Jr., M.T.
      ,
      • Mossing M.C.
      • Record Jr., M.T.
      ,
      • Whitson P.A.
      • Olson J.S.
      • Matthews K.S.
      ,
      • Brenowitz M.
      • Jamison E.
      ,
      • Brenowitz M.
      • Pickar A.
      • Jamison E.
      ,
      • Kramer H.
      • Niemoller M.
      • Amouyal M.
      • Revet B.
      • von Wilcken-Bergmann B.
      • Müller-Hill B.
      ,
      • Bellomy G.R.
      • Mossing M.C.
      • Record Jr., M.T.
      ). 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 (
      • Fried M.G.
      • Crothers D.M.
      ,
      • Ruusala T.
      • Crothers D.M.
      ). 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 LacI adi were conducted to compare directly the kinetic properties of this dimeric form of LacI with the tetramer (
      • Ruusala T.
      • Crothers D.M.
      ,
      • Fickert R.
      • Müller-Hill B.
      ). 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 (
      • Riggs A.D.
      • Bourgeois S.
      • Cohn M.
      ,
      • Berg O.G.
      • Winter R.B.
      • von Hippel P.H.
      ,
      • Winter R.B.
      • von Hippel P.H.
      ,
      • Barkley M.D.
      ,
      • Winter R.B.
      • Berg O.G.
      • von Hippel P.H.
      ) used LacI concentrations on the order of 10−12m. The more recent comparisons of DNA binding by LacI and LacI adi utilized protein concentrations as high as 10−9m (
      • Ruusala T.
      • Crothers D.M.
      ,
      • Fickert R.
      • Müller-Hill B.
      ), well below indirect estimates of 10−8–10−12m for the Kd of the LacI dimer-tetramer equilibrium (
      • Fickert R.
      • Müller-Hill B.
      ,
      • Brenowitz M.
      • Pickar A.
      • Jamison E.
      ,
      • Royer C.A.
      • Chakerian A.E.
      • Matthews K.S.
      ). However, a recent analysis of dimer-tetramer linkage with sequence-specific DNA binding suggests that LacI dimers are not present even at concentrations of 10−12m (
      • Levandoski M.M.
      • Tsodikov O.V.
      • Frank D.E.
      • Melcher S.E.
      • Saecker R.M.
      • Record Jr., M.T.
      ). In addition to eliminating dimer-to-tetramer association, the deletion of the COOH-terminal residues of LacI which create the LacI adi mutant also weakens the monomer-dimer association (
      • Brenowitz M.
      • Mandal N.
      • Pickar A.
      • Jamison E.
      • Adhya S.
      ). A Kd of 7.7 × 10−8m was determined for this equilibrium (
      • Chen J.
      • Matthews K.S.
      ). Thus, the previous studies (
      • Ruusala T.
      • Crothers D.M.
      ,
      • Fickert R.
      • Müller-Hill B.
      ) 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 LacI adi concentrations where their oligomeric states were well defined. For LacI, concentrations in the range of 6.3–25 × 10−9m 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 (
      • Brenowitz M.
      • Jamison E.
      ,
      • Brenowitz M.
      • Pickar A.
      • Jamison E.
      ). LacI adi is dimeric in the concentration range of 25–100 × 10−9m employed in these studies. In addition, high protein concentrations were required to saturate the operator with LacI adi since the linked monomer-dimer equilibrium results in diminished apparent binding affinity (
      • Brenowitz M.
      • Mandal N.
      • Pickar A.
      • Jamison E.
      • Adhya S.
      ,
      • Chen J.
      • Matthews K.S.
      ).
      The data presented in Figs. 3 and 4 confirm the differences inka and reveal different salt dependences for the dimeric LacI adi 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 (
      • Winter R.B.
      • Berg O.G.
      • von Hippel P.H.
      ). A comparison of rates between the past (
      • Winter R.B.
      • Berg O.G.
      • von Hippel P.H.
      ) 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 (107–108m−1 s−1; 2, 7) at 200 mm KCl (Fig. 3). The independence ofka for the tetramer over this [KCl] range was unexpected. The model of Berg et al. (
      • Berg O.G.
      • Winter R.B.
      • von Hippel P.H.
      ) 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.
      • Berg O.G.
      • Winter R.B.
      • von Hippel P.H.
      for the simulations described below. The value for the nonspecific binding constant,KRD , was calculated from Ref.
      • Ha J.-H.
      • Capp M.W.
      • Hohenwalter M.D.
      • Baskerville M.
      • Record Jr., M.T.
      . 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 ofka determined for LacI could be described by superimposing a direct transfer rate constant (ν, s−1) upon the linear diffusion rate constant (D1, cm2 s−1) determined from LacI adi data. Because of the high correlation between ν and D1, it is not possible to determine both constants simultaneously. Thus, the assumption was made that LacI adi binding is facilitated solely by sliding. In the absence of direct transfer (ν = 0), the values of ka determined for LacI adi as a function of [KCl] and DNA length are described by values of D1 ranging from 3 × 10−14 to 2 × 10−13cm2 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 (ν = 10 and 1,000 transfers s−1, the latter value being theoretical upper limit for ν; 2), are superimposed upon the value ofD1 of 3 × 10−14cm2 s−1 which describes the [KCl] dependence of binding of the dimeric LacI adi. This combination of sliding and direct transfer accounts for the rate enhancement of LacI relative to LacI adi 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 ν alone does not account for the relative difference in association rates observed between LacI and LacI adi (Fig. 3).
      Simulations of the LacI data with D1 ranging from 3 × 10−10 to 7 × 10−11cm2 s−1 and ν = 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 ν andD1, an increase in D1appears to be a necessary component of LacI rate enhancement relative to LacI adi. Because of the high theoretical correlation between ν 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 LacI adi 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 LacI adi (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 LacI adi 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 LacI adiprotein (
      • Stickle D.F.
      • Liu G.
      • Fried M.G.
      ). 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 (
      • Stickle D.F.
      • Liu G.
      • Fried M.G.
      ), 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 (
      • Lewis M.
      • Chang G.
      • Horton N.C.
      • Kercher M.A.
      • Pace H.C.
      • Schumacher M.A.
      • Brennan R.G.
      • Lu P.
      ) 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 (
      • Lewis M.
      • Chang G.
      • Horton N.C.
      • Kercher M.A.
      • Pace H.C.
      • Schumacher M.A.
      • Brennan R.G.
      • Lu P.
      ). 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 (
      • Levandoski M.M.
      • Tsodikov O.V.
      • Frank D.E.
      • Melcher S.E.
      • Saecker R.M.
      • Record Jr., M.T.
      ). 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 LacI adi 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.

      REFERENCES

        • Riggs A.D.
        • Bourgeois S.
        • Cohn M.
        J. Mol. Biol. 1970; 53: 401-417
        • Berg O.G.
        • Winter R.B.
        • von Hippel P.H.
        Biochemistry. 1981; 20: 6929-6948
        • Winter R.B.
        • von Hippel P.H.
        Biochemistry. 1981; 20: 6948-6960
        • Barkley M.D.
        Biochemistry. 1981; 20: 3833-3842
        • Berg O.G.
        • Winter R.B.
        • von Hippel P.H.
        Trends Biochem. Sci. 1982; 7: 52-55
        • Berg O.G.
        • von Hippel P.H.
        Annu. Rev. Biophys. Biophys. Chem. 1985; 14: 131-160
        • Berg O.G.
        • von Hippel P.H.
        Trends Biochem. Sci. 1988; 13: 207-211
        • von Hippel P.H.
        • Berg O.G.
        J. Biol. Chem. 1989; 264: 675-678
        • Mazur S.J.
        • Record Jr., M.T.
        Biopolymers. 1989; 28: 929-953
        • Kim J.G.
        • Takeda Y.
        • Matthews B.W.
        • Anderson W.F.
        J. Mol. Biol. 1987; 196: 149-158
        • Jack W.E.
        • Terry B.J.
        • Modrich P.
        Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4010-4014
        • Terry B.J.
        • Jack W.E.
        • Modrich P.
        J. Biol. Chem. 1985; 260: 13130-13137
        • Kabata H.
        • Kurosawa O.
        • Arai I.
        • Washizu M.
        • Margarson S.A.
        • Glass R.E.
        • Shimamoto N.
        Science. 1993; 262: 1561-1563
        • Bresloff J.L.
        • Crothers D.M.
        J. Mol. Biol. 1975; 95: 103-123
        • Fried M.G.
        • Crothers D.M.
        J. Mol. Biol. 1984; 172: 263-282
        • Mossing M.C.
        • Record Jr., M.T.
        J. Mol. Biol. 1985; 186: 295-305
        • Mossing M.C.
        • Record Jr., M.T.
        Science. 1986; 233: 889-892
        • Whitson P.A.
        • Olson J.S.
        • Matthews K.S.
        Biochemistry. 1986; 25: 3852-3858
        • Ruusala T.
        • Crothers D.M.
        Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4903-4907
        • Fickert R.
        • Müller-Hill B.
        J. Mol. Biol. 1992; 226: 59-68
        • Hsieh M.
        • Brenowitz M.
        Methods Enzymol. 1996; 274: 478-492
        • Hsieh M.
        • Hensley P.
        • Brenowitz M.
        • Fetrow J.S.
        J. Biol. Chem. 1994; 269: 13825-13835
        • Weickert M.J.
        • Adhya S.
        J. Biol. Chem. 1992; 267: 15869-15874
        • Nichols J.C.
        • Vyas N.K.
        • Quiocho F.A.
        • Matthews K.S.
        J. Biol. Chem. 1993; 268: 17602-17612
        • Schumacher M.A.
        • Choi K.Y.
        • Zalkin H.
        • Brennan R.G.
        Science. 1994; 266: 763-770
        • Friedman A.M.
        • Fischmann T.O.
        • Steitz T.A.
        Science. 1995; 268: 1721-1727
        • Sclavi B.
        • Woodson S.
        • Sullivan M.
        • Chance M.
        • Brenowitz M.
        J. Mol. Biol. 1997; 266: 144-159
        • Brenowitz M.
        • Jamison E.
        Biochemistry. 1993; 32: 8693-8701
      1. Dalma-Weiszhausz, D. D. (1995) The Escherichia coli gal Operon: Cross-talk between Positive and Negative Regulation of Transcription. Ph.D. thesis, pp. 40–54, Albert Einstein College of Medicine, New York.

        • Dalma-Weiszhausz D.D.
        • Brenowitz M.
        Biochemistry. 1996; 35: 3735-3745
        • Brenowitz M.
        • Senear D.F.
        • Shea M.A.
        • Ackers G.K.
        Methods Enzymol. 1986; 130: 132-181
        • Brenowitz M.
        • Senear D.F.
        Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, New York1989: 12.4.1-12.4.16
        • Brenowitz M.
        • Senear D.F.
        • Jamison L.
        • Dalma-Weiszhausz D.D.
        Revzin A. Footprinting Techniques for Studying Nucleic Acid-Protein Complexes: Separation, Detection, and Characterization of Biological Macromolecules. Academic Press, New York1993: 1-43
        • Winter R.B.
        • Berg O.G.
        • von Hippel P.H.
        Biochemistry. 1981; 20: 6961-6977
        • Johnson M.L.
        • Faunt L.M.
        Methods Enzymol. 1992; 210: 1-37
        • Record M.T.
        • deHaseth P.L.
        • Lohman T.M.
        Biochemistry. 1977; 16: 4791-4796
        • Brenowitz M.
        • Pickar A.
        • Jamison E.
        Biochemistry. 1991; 30: 5986-5998
        • Brenowitz M.
        • Mandal N.
        • Pickar A.
        • Jamison E.
        • Adhya S.
        J. Biol. Chem. 1991; 266: 1281-1288
        • Chen J.
        • Matthews K.S.
        Biochemistry. 1994; 26: 8728-8735
        • Garner M.M.
        • Rau D.C.
        EMBO J. 1995; 14: 1257-1263
        • Stickle D.F.
        • Liu G.
        • Fried M.G.
        Eur. J. Biochem. 1994; 226: 869-876
        • Vossen K.M.
        • Stickle D.F.
        • Fried M.G.
        J. Mol. Biol. 1996; 255: 44-54
        • Khoury A.M.
        • Lee H.J.
        • Lillis M.
        • Lu P.
        Biochim. Biophys. Acta. 1990; 1087: 55-60
        • Jeltsch A.
        • Alves J.
        • Wolfes H.
        • Maass G.
        • Pingoud A.
        Biochemistry. 1994; 33: 10216-10219
        • Kramer H.
        • Niemoller M.
        • Amouyal M.
        • Revet B.
        • von Wilcken-Bergmann B.
        • Müller-Hill B.
        EMBO J. 1987; 6: 1481-1491
        • Bellomy G.R.
        • Mossing M.C.
        • Record Jr., M.T.
        Biochemistry. 1988; 27: 3900-3906
        • Royer C.A.
        • Chakerian A.E.
        • Matthews K.S.
        Biochemistry. 1990; 29: 4959-4966
        • Levandoski M.M.
        • Tsodikov O.V.
        • Frank D.E.
        • Melcher S.E.
        • Saecker R.M.
        • Record Jr., M.T.
        J. Mol. Biol. 1996; 260: 697-717
        • Ha J.-H.
        • Capp M.W.
        • Hohenwalter M.D.
        • Baskerville M.
        • Record Jr., M.T.
        J. Mol. Biol. 1992; 228: 252-264
        • Lewis M.
        • Chang G.
        • Horton N.C.
        • Kercher M.A.
        • Pace H.C.
        • Schumacher M.A.
        • Brennan R.G.
        • Lu P.
        Science. 1996; 271: 1247-1254