Cooperative and Anticooperative Effects in Binding of the First and Second Plasmid OsymOperators to a LacI Tetramer: Evidence for Contributions of Non-operator DNA Binding by Wrapping and Looping

The interaction oflacoperator DNA withlacrepressor (LacI) is a classic example of a genetic regulatory switch. To dissect the role of stoichiometry, subunit association, and effects of DNA length in positioning this switch, we have determined binding isotherms for the interaction of LacI with a high affinity (O^sym) operator on linearized plasmid (2500 bp) DNA over a wide range of macromolecular concentrations (10⁻¹⁴to 10⁻⁸M). Binding data were analyzed using a thermodynamic model involving four equilibria: dissociation of tetramers (T) into dimers (D), and binding of operator-containing plasmid DNA (O) to dimers and tetramers to form three distinct complexes, DO, TO, and TO₂. Over the range of con- centrations of repressor, operator, and salt (0.075 M K⁺to 0.40 M K⁺) investigated, we find no evidence for any significant thermodynamic effect of LacI dimers. Instead, all isotherms can be interpreted in terms of just two equilibria, involving only T and the TO and TO₂complexes. As a reference binding equilibrium, which we propose must approximate the DO binding interaction, we compare the plasmid O^symresults with our extensive studies of the binding of a 40 bp O^symDNA fragment to LacI. On this basis, we obtain a lower bound on the LacI dimer – tetramer equilibrium constant and values of the equilibrium constants for formation of TO and TO₂complexes.At a salt concentration of 0.40 M, the O^symplasmid binding data are consistent with a model with two independent and identical binding sites for operator per LacI tetramer, in which the binding to a site on the tetramer is only slightly more favorable than the reference binding interaction. Increasingly large deviations from the independent-site model are observed as the salt concentration is reduced; binding of a second operator to form TO₂becomes strongly disfavored relative to formation of TO at low salt concentrations (0.075 to 0.125 M). In addition, binding of both the first and second plasmid operator DNA molecules to the tetramer becomes increasingly more favorable than the reference binding interaction as [K⁺] is reduced from 0.40 M to 0.125 M. At 0.075 M K⁺, however, the strength of binding of the second plasmid operator DNA to the LacI tetramer is dramatically reduced; this interaction is much less favorable than binding the first plasmid operator DNA, and becomes much less favorable than the reference binding interaction. We propose that these differences arise from changes in the nature of the TO and TO₂complexes with decreasing salt concentration. At low salt concentration, we suggest the hypothesis that flanking non-operator sequences bind non-specifically (coulombically) by local wrapping, and that distant regions of non-operator DNA occupy the second operator-binding site by looping. We propose that wrapping stabilizes both 1:1 and 2:1 complexes at low salt concentration, and that looping stabilizes the 1:1 complex but competitively destabilizes the 2:1 TO₂complex at low salt concentration. These effects must play a role in adjusting the stability and structure of the LacI-lac operator repression complex as the cytoplasmic [K⁺] varies in response to changes in extracellular osmolarity.     

Sequence-specific interactions of the tight-binding I12-X86 lac repressor with non-operator DNA.

The tight-binding I12-X86 lac repressor binds to non-operator DNA in a sequence-specific fashion. Using the DNA of the E. coli I gene we have investigated these sequence-specific interactions and compared them to the operator binding of wild-type repressor. The specific, non-operator DNA interactions are sensitive to the inducer IPTG. One strong binding site in the I gene DNA was found to be one of two expected on the basis of their homology with the lac operator. The binding of I12-X86 repressor to this site was visualized using the footprinting technique, and found to be consistent with an operator-like binding configuration. The protection pattern extends into an adjacent sequence suggesting that two repressor tetramers are bound in tandem.     

Thermodynamic stoichiometries of participation of water, cations and anions in specific and non-specific binding of lac repressor to DNA. Possible thermodynamic origins of the "glutamate effect" on protein-DNA interactions.

The objective of this study is to quantify the contributions of cations, anions and water to stability and specificity of the interaction of lac repressor (lac R) protein with the strong-binding symmetric lac operator (Osym) DNA site. To this end, binding constants Kobs and their power dependences on univalent salt (MX) concentration (SKobs = d log Kobs/d log[MX]) have been determined for the interactions of lac R with Osym operator and with non-operator DNA using filter binding and DNA cellulose chromatography, respectively. For both specific and non-specific binding of lac R, Kobs at fixed salt concentration [KX] increases when chloride (Cl-) is replaced by the physiological anion glutamate (Glu-). At 0.25 M-KX, the increase in Kobs for Osym is observed to be approximately 40-fold, whereas for non-operator DNA the increase in Kobs is estimated by extrapolation to be approximately 300-fold. For non-operator DNA, SKobsRD is independent of salt concentration within experimental uncertainty, and is similar in KCl (SKobs,RDKCl = -9.8(+/- 1.0) between 0.13 M and 0.18 M-KCl) and KGlu (SKobs,RDKGlu = -9.3(+/- 0.7) between 0.23 M and 0.36 M-KGlu). For Osym DNA, SKobsRO varies significantly with the nature of the anion, and, at least in KGlu appears to decrease in magnitude with increasing [KGlu]. Average magnitudes of SKobsRO are less than SKobsRD, and, for specific binding decrease in the order [SKobsRO,KCl[>[SKobsRO,KAc[>[SKobsRO,KGlu[ . Neither KobsRO nor SKobsRO is affected by the choice of univalent cation M+ (Na+, K+, NH4+, or mixtures thereof, all as the chloride salt), and SKobsRO is independent of [MCl] in the range examined (0.125 to 0.3 M). This behavior of SKobsRO is consistent with that expected for a binding process with a large contribution from the polyelectrolyte effect. However, the lack of an effect of the nature of the cation on the magnitude of KobsRO at a fixed [MX] is somewhat unexpected, in view of the order of preference of cations for the immediate vicinity of DNA (NH4+ > K+ > Na+) observed by 23Na nuclear magnetic resonance. For both specific and non-specific binding, the large stoichiometry of cation release from the DNA polyelectrolyte is the dominant contribution to SKobs. To interpret these data, we propose that Glu- is an inert anion, whereas Ac- and Cl- compete with DNA phosphate groups in binding to lac repressor. A thermodynamic estimate of the minimum stoichiometry of water release from lac repressor and Osym operator (210(+/- 30) H2O) is determined from analysis of the apparently significant reduction in [SKobsRO,KGlu[ with increasing [KGlu] in the range 0.25 to 0.9 M. According to this analysis, SKobs values of specific and non-specific binding in KGlu differ primarily because of the release of water in specific binding. In KAc and KCl, we deduce that anion competition affects Kobs and SKobs to an extent which differs for different anions and for the different binding modes.     

Binding of E.coli lac repressor to non-operator DNA*

It is shown by melting profile analysis of lac repressor-DNA complexes that repressor binds tightly and preferentially (relative to single-stranded DNA) to double-stranded non-operator DNA. This binding stabilizes the DNA against melting and the repressor against thermal denaturation. Analysis of the extent of stabilization and the rate of dissociation of repressor from non-operator DNA as a function of sodium ion concentration shows, in confirmation of other studies,(3,4) that the binding constant (K(RD)) is very ionic strength dependent; K(RD) increases from approximately 10(6) M(-1) at approximately 0.1 M Na(+) to values in excess of 10(10) M(-1) at 0.002 M Na(+). Repressor bound to non-operator DNA is not further stabilized against thermal denaturation by inducer binding, indicating that the inducer and DNA binding sites probably represent separately stabilized local conformations. Transfer melting experiments are used to measure the rate of dissociation of repressor from operator DNA. These experiments show that most of the ionic strength dependence of the binding constant is in the dissociation process; the estimated dissociation rate constant decreases from greater than 10(-1) sec(-1) at [Na(+)] >/= 0.02 M to less than 10(-4) sec(-1) at [Na(+)] 相似文献   

Interaction of mutant lambda repressors with operator and non-operator DNA

We have described a set of mutations that alter side-chains on the operator binding surface of lambda repressor. In this paper, we study the interactions of 12 purified mutant repressors with operator and non-operator DNA. The mutant proteins have operator affinities that are reduced from tenfold to greater than 10,000-fold compared to wild-type. Nine of the mutants have affinities for non-operator DNA that are similar to wild-type, two mutants show decreased non-specific binding, and one mutant has increased affinity for non-operator DNA. We discuss these findings in terms of the structural and energetic contributions of side-chain--DNA interactions, and show that certain contacts between the repressor and the operator backbone contribute both energy and specificity to the interaction.     

TET repressor.tet operator complex formation induces conformational changes in the tet operator DNA.

The structural changes of the tet operator DNA upon binding of the TET repressor protein are examined by circular dichroism. For this purpose a 70 bp DNA fragment was prepared which contains both tet operators. About 67% of the base pairs of this DNA are involved in specific interaction with the TET repressor. A rather large change in the CD of the DNA is induced by binding of the TET repressor. The shape of the CD difference spectrum is similar to the respective difference found for the lac operator DNA upon complex formation with the lac repressor. However, the effect induced by the TET repressor on tet operator DNA seems to comprise both the specific and non-specific effect of the lac repressor on the structure of DNA [Culard, F. and Maurizot, J.C. (1981) Nucl. Acids Res. 9, 5157-5184]. Specificity of binding is confirmed by the lack of any effect of the TET repressor on the CD of a 95 bp lac operator containing DNA fragment, by the reduced mobility of TET repressor.tet operator complexes on polyacrylamide gels under CD conditions, and by a titration experiment of tet operator DNA with TET repressor employing the CD change. The latter experiment reveals a stoichiometry of four TET repressors per tet operon control region.     

Site-specific DNA-affinity chromatography of the lac repressor.

To test the feasibility of site-specific DNA-affinity chromatography, E. coli lac repressor was bound to an operator-containing DNA column, and in parallel to a non-operator DNA column. Salt gradient elution shows: 1) elution from non-operator DNA was near 250mM KCl or NaCl; interpretation of this result suggests the usefulness of the procedure for studying salt-dependence of DNA-protein affinities; 2) elution from operator-containing DNA was delayed (average elution = 1000mM salt), demonstrating a feasibility of site-specific DNA-affinity chromatography, if one provides a sufficiently favorable ratio of specific to non-specific DNA binding sites; 3) repressor eluted from operator-containing DNA over a very broad salt range, which may represent chromatography-generated repressor heterogeneity.     

Interaction between the lac operator and the lac repressor headpiece: fluorescence and circular dichroism studies

The interaction between the lac repressor headpiece and a small operator DNA fragment has been examined by fluorescence and circular dichroism (c.d.) measurements. Binding of the headpiece to the DNA fragment induces a strong quenching of the fluorescence of its tyrosine residues. Quantitative analysis of the fluorescence data demonstrates that, in a first step, two headpieces bind very strongly to the DNA fragment then weaker binding occurs. C.d. demonstrates that the binding induces conformational changes of the DNA. The c.d. change produced upon binding of the first two headpieces differs from that induced upon binding of two further headpieces . Binding of the second pair of headpieces is similar to non-specific binding to non-operator DNA. The conformation of the operator DNA in the presence of two headpieces differs drastically from that in presence of lac repressor. Addition of the core to the lac operator does not induce any conformational change of the nucleic acids. These results are discussed with respect to the relative roles of core and headpieces in the lac repressor-lac operator interaction.     

Isolation and characterization of LexA mutant repressors with enhanced DNA binding affinity.

The LexA repressor from Escherichia coli is a sequence-specific DNA binding protein that shows no pronounced sequence homology with any of the known structural motifs involved in DNA binding. Since little is known about how this protein interacts with DNA, we have selected and characterized a great number of intragenic, second-site mutations which restored at least partially the activity of LexA mutant repressors deficient in DNA binding. In 47 cases, the suppressor effect of these mutations was due to an Ind- phenotype leading presumably to a stabilization of the mutant protein. With one exception, these second-site mutations are all found in a small cluster (amino acid residues 80 to 85) including the LexA cleavage site between amino acid residues 84 and 85 and include both already known Ind- mutations as well as new variants like GN80, GS80, VL82 and AV84. The remaining 26 independently isolated second-site suppressor mutations all mapped within the amino-terminal DNA binding domain of LexA, at positions 22 (situated in the turn between helix 1 and helix 2) and positions 57, 59, 62, 71 and 73. These latter amino acid residues are all found beyond helix 3, in a region where we have previously identified a cluster of LexA (Def) mutant repressors. In several cases the parental LexA (Def) mutation has been removed by subcloning or site-directed mutagenesis. With one exception, these LexA variants show tighter in vivo repression than the LexA wild-type repressor. The most strongly improved variant (LexA EK71, i.e. Glu71----Lys) that shows an about threefold increased repression rate in vivo, was purified and its binding to a short consensus operator DNA fragment studied using a modified nitrocellulose filter binding assay. As expected from the in vivo data, LexA EK71 interacts more tightly with both operator and (more dramatically) with non-operator DNA. A determination of the equilibrium association constants of LexA EK71 and LexA wild-type as a function of monovalent salt concentration suggests that LexA EK71 might form an additional ionic interaction with operator DNA as compared to the LexA wild-type repressor. A comparison of the binding of LexA to a non-operator DNA fragment further shows that LexA interacts with the consensus operator very selectively with a specificity factor of Ks/Kns of 1.4 x 10(6) under near-physiological salt conditions.     

Hinge-helix formation and DNA bending in various lac repressor-operator complexes

The hinge-region of the lac repressor plays an important role in the models for induction and DNA looping in the lac operon. When lac repressor is bound to a tight-binding symmetric operator, this region forms an alpha-helix that induces bending of the operator. The presence of the hinge-helices is questioned by previous data that suggest that the repressor does not bend the wild-type operator. We show that in the wild-type complex the hinge-helices are formed and the DNA is bent, similar to the symmetric complex. Furthermore, our data show differences in the binding of the DNA binding domains to the half-sites of the wild-type operator and reveal the role of the central base-pair of the wild-type operator in the repressor-operator interaction. The differences in binding to the operator half-sites are incorporated into a model that explains the relative affinities of the repressor for various lac operator sequences that contain left and right half-sites with different spacer lengths.     

lac repressor forms loops with linear DNA carrying two suitably spaced lac operators.

Tetrameric lac repressor may bind to two lac operators on one DNA fragment and induce the intervening DNA to form a loop. Electron microscopy, non-denaturing polyacrylamide gel electrophoresis, and DNase I protection experiments were used to demonstrate such DNA loops, where the distance between the centres of symmetry of the two lac operators varies between 63 and 535 bp. Formation of a DNA loop is favoured by correct phasing of the two lac operators and a low concentration of both components of the reaction. When a large excess of lac repressor over DNA is used, a 'tandem' structure is observed, in which both lac operators are occupied independently by two repressor tetramers. When the concentrations of both lac repressor and lac operator are high, a 'sandwich' structure is observed, in which two DNA molecules are connected by two lac repressor tetramers in trans.     

Preferential protection of the minor groove of non-operator DNA by lac repressor against methylation by dimethyl sulphate.

The binding of lactose repressor to non-operator DNA was studied by the modification of several DNA's, including glycosylated DNA, with dimethyl sulphate, which affects the minor and major grooves of DNA and single stranded DNA regions. The non-specific binding of the repressor to DNA protected the minor groove but apparently not the major groove of the DNA double helix against methylation and did not increase the content of single stranded DNA regions. This suggests that the repressor on binding to non-operator DNA makes contacts mainly in the minor groove of DNA and does not uncoil the DNA double helix. This is different from the interaction of the repressor with lactose operator DNA which occurs, as shown by Gilbert et al. (1), along both the major and the minor groove.     

Role of hydration in the binding of lac repressor to DNA

The osmotic stress technique was used to measure changes in macromolecular hydration that accompany binding of wild-type Escherichia coli lactose (lac) repressor to its regulatory site (operator O1) in the lac promoter and its transfer from site O1 to nonspecific DNA. Binding at O1 is accompanied by the net release of 260 +/- 32 water molecules. If all are released from macromolecular surfaces, this result is consistent with a net reduction of solvent-accessible surface area of 2370 +/- 550 A. This area is only slightly smaller than the macromolecular interface calculated for a crystalline repressor dimer-O1 complex but is significantly smaller than that for the corresponding complex with the symmetrical optimized O(sym) operator. The transfer of repressor from site O1 to nonspecific DNA is accompanied by the net uptake of 93 +/- 10 water molecules. Together these results imply that formation of a nonspecific complex is accompanied by the net release of 165 +/- 43 water molecules. The enhanced stabilities of repressor-DNA complexes with increasing osmolality may contribute to the ability of Escherichia coli cells to tolerate dehydration and/or high external salt concentrations.     

Probing co-operative DNA-binding in vivo. The lac O1:O3 interaction

The lac primary (O1) and weak upstream pseudo (O3) operators contained on a plasmid were footprinted in vivo in order to determine whether they act co-operatively in binding lac repressor in the cell. The occupancy at O3 by lac repressor was substantially reduced upon deletion of the lac primary operator, demonstrating co-operativity at a distance. Plots of operator occupancy versus active repressor concentration were obtained for each operator by treating the cells with different amounts of the lac inducer isopropyl-beta-D-thiogalactoside and probing lac repressor binding. This analysis can be used to obtain relative binding constants in vivo and demonstrates that O3 binds repressor only 10.3-fold less tightly than O1 in their co-operative interaction. The removal of DNA torsional tension in vivo by the use of coumermycin leads to the same loss of binding at O3 as does deleting O1. These in-vivo results are analogous to the in-vitro situation, where O3 binds repressor strongly in a DNA repression loop only on supercoiled templates.     

Isolation of amino-terminal fragment of lactose repressor necessary for DNA binding.

lac repressor can be dissected by trypsin into a homogenous tetrameric core (accounting for residues 60 to 347), carrying inducer binding activity, and the monomeric amino-terminal peptides ("headpieces") accounting for residues 1 to 59 and 1 to 51, respectively. This restriction of the action of trypsin on lac repressor is obtained in 1 M Tris-HCl (pH 7.5)-30% in glycerol at 25 degrees C since only the peptide bonds at lysine-59 and to a lesser extent after at arginine-51 are cleaved under these conditions. The headpieces can be purified by gel filtration. They have ordered secondary structure as revealed by circular dichroism studies. The monomeric headpieces show the relatively weak binding to nonoperator DNA but not the highly specific and strong binding to operator DNA typical for tetrameric lac repressor.     

The interaction of RNA polymerase and lac repressor with the lac control region.

We have examined the interactions of lac repressor and RNA polymerase with the DNA of the lac control region, using a method for direct visualization of the regions of DNA protected by proteins from DNAase attack. The repressor protects the operator essentially as reported by Gilbert and Maxam (1) with some small modifications. However, the evidence reported here concerning the binding of RNA polymerase to the DNA of the promoter mutant UV5 indicates that : 1) the RNA polymerase molecule binds asymmetrically to the promoter DNA, 2) RNA polymerase protects DNA sequences to within a few bases of the CAP binding site, suggesting direct interaction between polymerase and the CAP protein at this site, 3) RNA polymerase still binds to the promoter when repressor is bound to the operator, but fails to form the same extensive complex.     

Energetic differences between the specific binding of a 40 bp DNA duplex and the lac promoter to lac repressor protein

The energetics of LRP binding to a 104 bp lac promoter determined from ITC measurements were compared to the energetics of binding to a shorter 40 bp DNA duplex with the 21 bp promoter binding site sequence. The promoter binding affinity of 2.47 +/- 0.0 1x 10(7) M(-1) was higher than the DNA binding affinity of 1.81 +/- 0.67 x 10(7) M(-1) while the binding enthalpy of -804 +/- 41 kJ mol(-1) was lower than that of the DNA binding enthalpy of -145 +/- 16 kJ mol(-1) at 298.15 K. Both the promoter and DNA binding reactions were exothermic in phosphate buffer but endothermic in Tris buffer that showed the transfer of four protons to LRP in the former reaction but only two in the latter. A more complicated dependence of these parameters on temperature was observed for promoter binding. These energetic differences are attributable to additional LRP-promoter interactions from wrapping of the promoter around the LRP.