Association kinetics with coupled diffusion: III. Ionic-strength dependence of the lac repressor-operator association

The repressor- operator association is treated in a model where the represser molecule can find its specific binding site the operator, on a large DNA chain by performing a one-dimensional diffusion along the chain. The ionic-strength depen deuce is calculated by introducing a screened electrostatic potential around the DNA chain and coupling the free diffusion of the represser in this potential to the proposed one-dimensional diffusion along the chain. The main influence on the association rate comes from the competitive binding of ions to the unspecific DNA sites. It is also demonstrated that during the time that the represser is bound in a global sense, the diffusion along the chain will be made up of a strictly one-dimensional motion over fairly short distances, interspersed with many local dissociations during which the repressor in essence is free in solution.     

Lac repressor-operator interaction: DNA length dependence

The interaction of the E. coli lac operon repressor with its operator DNA has been directly examined as a function of the length of operator-containing DNA. The apparent bimolecular association rate constants were calculated as ka = (kd/KD), where the dissociation equilibrium constant, KD and the dissociation rate constant, kd, were measured by nitrocellulose filter adsorption assays. The values obtained for the overall association rate constants are compared with theoretical association rate curves for specific mechanisms. Association of the repressor with short operator containing DNA fragments (less than 70 base pairs) occurs at rates expected of three-dimensional diffusion. Our data also imply that at longer DNA lengths a combination of three-dimensional diffusion with one-dimensional sliding along with hopping and/or intersegment transfer must be involved to facilitate the repressor operator association.     

Sliding and target location of DNA-binding proteins:an NMR view of the lac repressor system

In non-specific lac headpiece-DNA complexes selective NMR line broadening is observed that strongly depends on length and composition of the DNA fragments. This broadening involves amide protons found in the non-specific lac-DNA structure to be interacting with the DNA phosphate backbone, and can be ascribed to DNA sliding of the protein along the DNA. This NMR exchange broadening has been used to estimate the 1D diffusion constant for sliding along non-specific DNA. The observed 1D diffusion constant of 4×10^?12 cm²/s is two orders of magnitude smaller than derived from previous kinetic experiments, but falls in the range of values determined more recently using single molecule methods. This strongly supports the notion that sliding could play at most a minor role in the association kinetics of binding of lac repressor to lac operator and that other processes such as hopping and intersegment transfer contribute to facilitate the DNA recognition process.     

lac repressor-operator interaction. Analysis of the X86 repressor mutant

In vitro measurements show that the X86 repressor, which has an increased affinity for the lac operator as compared to wild-type repressor, also has an increased affinity for non-operator sites on Escherichia coli DNA. The rate constant of association of repressor and operator is decreased by E. coli DNA fivefold more for X86 repressor than for wild-type repressor. Low inducer concentrations increase the rate of association of X86 repressor and operator in the presence of E. coli DNA. In a partial equilibrium situation where part of the X86 repressor is bound to the operator, and part to either non-operator sites on E. coli DNA or to an O^c operator, the formation of complexes between X86 repressor and wild-type operator is favored by low inducer concentrations. Repression of the lac enzymes increases drastically in the X86 mutant in the absence of DNA synthesis in vivo. A new explanation for the in vivo characteristics of the X86 mutant is suggested.     

"Second" and "third operator" of the lac operon: an investigation of their role in the regulatory mechanism.

The influence of the two operator-like regions lying within or near the lac regulatory region on the binding of lac repressor to lac operator has been investigated. λdlac phages deleted either for the “second operator” in the beginning of the Z gene or deleted for the “third operator” at the end of the I gene were constructed. In in vitro binding experiments it could be shown that the deletion of secondary repressor binding sites from the lac regulatory region does not significantly alter the stability of the repressor—operator complex. Measuring the rate constant of association of repressor with operator in the presence of a 150-fold excess of unspecific DNA, we observed a concentration-dependent effect of the unspecific DNA, although the ratio of operator to non-operator DNA was kept constant. A small effect of the secondary binding sites is seen on the rate of association of repressor with operator, indicating that the secondary binding sites might play a role in facilitating association of repressor with operator under _{in vivo} conditions.     

Mapping of I gene mutations which lead to repressors with increased affinity for lac operator

A genetic mapping system is used to locate mutations on the lac repressor gene (I) which lead to repressor proteins with an increased affinity for operator DNA. These tight binding repressors (I^tb) are of particular interest since their analysis should allow some conclusions on the mechanism of interaction between repressor and operator. I^tb mutations were found to map in two regions of the I gene. One is near the amino-terminal end, a region which has been shown to be essential for the DNA binding properties of the repressor. The other region in which I^tb mutations were mapped codes for approximately amino acids 255 to 295 of the repressor, a region which had so far not been considered to be essential for the DNA binding properties of the repressor protein.     

1H, 13C and 15N backbone and side chain resonance assignments of a Myxococcus xanthus anti-repressor with no known sequence homologues

The CarS antirepressor activates a photo-inducible promoter in Myxococcus xanthus by physically interacting with the CarA repressor and eliminating the latter’s binding to operator DNA. Interestingly, interactions with both CarS and operator are crucially dependent on the DNA recognition helix of the CarA winged-helix DNA-binding domain. The CarA–CarS and the CarA-operator interfaces therefore overlap, and CarS may have structural features that mimic operator DNA. CarS has no known sequence homologues and its Gly and Pro contents are unusually high. Here, we report ¹H, ¹³C and ¹⁵N backbone and side chain assignments of CarS1, an 86-residue truncated yet fully functional variant of CarS. Secondary structural elements inferred from these data differ from those predicted from sequence.     

DNA-binding site of lac repressor probed by dimethylsulfate methylation of lac operator.

In order to compare the structures of the DNA-binding sites on variants of the lac repressor, we have studied the influence of these variants on the dimethylsulfate methylation of the lac operator. Since a bound protein changes the availability of specific purines in the operator to this chemical attack, comparisons of the methylation patterns will show similarities or differences in the protein DNA contacts. We compared lac repressor, induced lac repressor (repressor bound to the gratuitous inducer isopropyl-β-d-thiogalactoside), mutant repressors having increased operator affinities (X86, I12 and the X86-I12 double mutant) and repressor peptides (long headpiece, residues 1 to 59 and short headpiece. residues 1 to 51). All of these repressors and repressor peptides exhibit the same general pattern of protection and enhancement in the operator; however, the short headpiece pattern differs most from that of the repressor while the induced repressor and the long headpiece show intermediate patterns that are strikingly similar to each other. The mutant repressors do not show an isopropyl-β-d-thiogalactoside effect but otherwise are almost indistinguishable from wild-type repressor. These results demonstrate that all molecules bind to the operator using basically the same protein-DNA contacts; they imply that (1) most and possibly all repressor contacts to operator lie within amino acids 1 to 51, (2) inducer weakens many contacts rather than totally disrupting one or even a few and (3) the tight-binding mutants do not make additional contacts to the DNA.These results are consistent with a model in which the amino-terminal portions of two repressor monomers make the DNA contacts. We show that one can understand the affinity of binding as related to the accuracy of the register of the two amino-terminal portions along the DNA. Furthermore, the action of inducer and the behaviour of the tight binding mutants can be accomodated within a two-state model in which the strongly or weakly binding states correspond to structures in which the amino-terminal regions are rigidly or loosely held with respect to each other.     

Mechanism of translocation of uracil-DNA glycosylase from Escherichia coli between distributed lesions

Uracil–DNA glycosylase (Ung) is a DNA repair enzyme that excises uracil bases from DNA, where they appear through deamination of cytosine or incorporation from a cellular dUTP pool. DNA repair enzymes often use one-dimensional diffusion along DNA to accelerate target search; however, this mechanism remains poorly investigated mechanistically. We used oligonucleotide substrates containing two uracil residues in defined positions to characterize one-dimensional search of DNA by Escherichia coli Ung. Mg²⁺ ions suppressed the search in double-stranded DNA to a higher extent than K⁺ likely due to tight binding of Mg²⁺ to DNA phosphates. Ung was able to efficiently overcome short single-stranded gaps within double-stranded DNA. Varying the distance between the lesions and fitting the data to a theoretical model of DNA random walk, we estimated the characteristic one-dimensional search distance of ∼100 nucleotides and translocation rate constant of ∼2 × 10⁶ s⁻¹.     

A model for the binding of lac repressor to the lac operator

A model is suggested for the lac repressor binding to the lac operator in which the repressor polypeptide chain sequences from Gly 14 to Ala 32 and from Ala 53 to Leu 71 are involved in specific interaction with operator DNA. A correspondence between the protein and DNA sequences is found which explains specificity of the repressor binding to the lac operator. The model can be extended to describe specific binding of other regulatory proteins to DNA.     

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.     

Lac repressor–operator interaction: N-terminal peptide backbone 1H and 15N chemical shifts upon complex formation with DNA

When the lac repressor tetramer is bound to its DNA operator, methylation protection shows the nearly symmetric operator half-sites are contacted asymmetrically. This asymmetric binding results from the DNA sequence/structure. The reported structure of lac repressor N-terminal fragment and an 11 base-pair operator left half-site provides no information concerning the effect of asymmetric binding, from left operator half-site to right half-site, upon the polypeptide backbone. We isolated uniformly ¹⁵N labeled 56 amino acid wild-type (HP56WT) and 64 residue mutant [Pro3>Tyr3] (HP64tyr3) lac repressor N-terminal DNA binding fragments for ¹H/¹⁵N NMR studies with the left and right operators separately. Spectral coincidence of these longer fragments, indicating structural similarity with a protease derived 51 amino acid fragment for which the amide correlations are assigned, allows for assignment of the common amide resonances. For both HP56WT and HP64tyr3, spectral overlap of the amide correlation peaks reveals the polypeptide backbones of the uncomplexed polypeptides are structurally similar. Likewise the complexes of the peptides to the 11 base-pair lac left operator half-site are similar. On the other hand, complexes of HP56WT and the left compared to the right lac operator half-site show different residues of the polypeptide are affected by binding different half-sites of the operator. Thus, the DNA sequence/structure transmits asymmetry to the polypeptide backbone of the interacting protein.     

Interaction of the trp repressor with trp operator DNA fragments

The interaction of the trp repressor with several trp operator DNA fragments has been examined by DNA gel retardation assays and by circular dichroism, in the absence and presence of the corepressor l-tryptophan. The holorepressor binds stoichiometrically to both the trpO and aroH operators, forming 1:1 complexes. In the presence of excess protein, additional complexes are formed with these operator fragments. The relative electrophoretic mobilities of the 1:1 complexes differ significantly for trp and aroH operators, indicating that they differ substantially in gross structure. A mutant trp operator, trpO ^c, has low affinity for the holorepressor, and forms only complexes with stoichiometries of 2:1 (repressor: DNA) or higher, which have a very low electrophoretic mobility. Specific binding is also accompanied by a large increase in the intensity of the near ultraviolet circular dichroism, with only a small blue shift, which is consistent with significant changes in the conformation of the DNA. Large changes in the chemical shifts of three resonances in the ³¹P NMR spectrum of both the trp operator and the aroH operator occur on adding repressor only in the presence of L-tryptophan, consistent with localised changes in the backbone conformation of the DNA.Abbreviations CD circular dichroism - trpO, trpR aroH trp operator fragments - trpO ^c trpMH mutant trp operator fragments     

Fluorescence Quenching Studies of Trp Repressor–Operator Interaction

Steady-state quenching and time-resolved fluorescence measurements of L-tryptophan binding to the tryptophan-free mutant W19/99F of the tryptophan repressor of Escherichia coli have been used to observe the coreperessor microenvirnment changes upon ligand binding. Using iodide and acrylamide as quenchers, we have resolved the emission spectra of the corepressor into two components. The bluer component of L-tryptophan buried in the holorepressor exhibits a maximum of the fluorescence emission at 336 nm and can be characterized by a Stern–Volmer quenching constant equal to about 2.0–2.3 M^?1. The second, redder component is exposed to the solvent and possesses the fluorescence emission and Stern–Volmer quenching constant characteristic of L-tryptophan in the solvent. When the Trp holorepressor is bound to the DNA operator, further alterations in the corepressor fluorescence are observed. Acrylamide quenching experiments indicate that the Stern–Volmer quenching constant of the buried component of the corepressor decreases drastically to a value of 0.56 M^?1. The fluorescence lifetimes of L-tryptophan in a complex with Trp repressor decrease substantially upon binding to DNA, which indicates a dynamic mechanism of the quenching process.