DNA supercoiling changes the spacing requirement of two lac operators for DNA loop formation with lac repressor.

We have used a gel retardation assay to investigate the influence of DNA supercoiling on loop formation between lac repressor and two lac operators. A series of 15 DNA minicircles of identical size (452 bp) was constructed carrying two lac operators at distances ranging from 153 to 168 bp. Low positive or negative supercoiling (sigma = +/- 0.023) changed the spacing between the two lac operators required for the formation of the most stable loops. This reveals the presence of altered double helical repeats (ranging from 10.3 to 10.7 bp) in supercoiled DNA minicircles. At elevated negative supercoiling (sigma = -0.046) extremely stable loops were formed at all operator distances tested, with a slight spacing periodicity remaining. After relaxation of minicircle-repressor complexes with topoisomerase I one superhelical turn was found to be constrained in those minicircles which carry operators at distances corresponding to a non-integral number of helical turns. This indicates that DNA loop formation can define local DNA domains with altered topological properties of the DNA helix.     

DNA supercoiling promotes formation of a bent repression loop in lac DNA

Titration experiments on supercoiled lac DNA show that one repressor tetramer can bind simultaneously to the primary lac operator and to the very weak lac pseudo-operator, located 93 base-pairs apart. The formation of this complex is accompanied by the appearance of an extreme hypersensitive site in a five base-pair sequence located approximately midway between the operators. This remote sequence is hypersensitive to attack by two different chemical probes, dimethyl sulfate and potassium permanganate, the latter of which is a new probe for distorted DNA. We interpret these results in terms of a complex in which lac repressor holds two remote operators together in a DNA loop. The formation of this bent DNA loop requires negative DNA supercoiling. In vivo, both lac operators bind repressor even though the presence of multiple operator copies has forced the two operators to compete for a limited amount of repressor. This suggests that the operator and pseudo-operator have similar affinities for repressor in vivo. Such similar affinities were observed in vitro only when DNA supercoiling forced formation of a repression loop.     

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.     

Quality and position of the three lac operators of E. coli define efficiency of repression.

Repression of the lac promoter may be achieved in two different ways: either by interference with the action of RNA polymerase or by interference with CAP activation. We investigated cooperative repression of the Escherichia coli lac operon by systematic conversion of its three natural operators (O1, O2 and O3) on the chromosome. We find that cooperative repression by tetrameric Lac repressor increases with both quality and proximity of the interacting operators. A short distance of 92 bp allows effective repression by two very weak operators (O3, O3). The cooperativity of lac operators is discussed in terms of a local increase of repressor concentration. This increase in concentration depends on flexible DNA which allows loop formation.     

How Lac repressor finds lac operator in vitro.

Filter-binding and gel mobility shift assays were used to analyse the kinetics of the interaction of Lac repressor with lac operator. A comparison of the two techniques reveals that filter-binding assays with tetrameric Lac repressor have often been misinterpreted. It has been assumed that all complexes of Lac repressor and lac operator DNA bind with equal affinity to nitrocellulose filters. This assumption is wrong. Sandwich or loop complexes where two lac operators bind to one tetrameric Lac repressor are not or are only badly retained on nitrocellulose filters under normal conditions. Taking this into account, dimeric and tetrameric Lac repressor do not show any DNA-length dependence of their association and dissociation rate constants when they bind to DNA fragments smaller than 2455 base-pairs carrying a single symmetric ideal lac operator. A ninefold increased association rate to ideal lac operator on lambda DNA is observed for tetrameric but not dimeric Lac repressor. It is presumably due to intersegment transfer involving lac operator-like sequences.     

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.     

Protein-induced unwinding of DNA: measurement by gel electrophoresis of complexes with DNA minicircles. Application to restriction endonuclease EcoRI, catabolite gene activator protein and lac repressor.

An electrophoretic procedure for the measurement of the helix unwinding induced by a sequence-specific protein is described. The method, which was applied here to EcoR I, CAP and lac repressor, involved the migration of the complexes with positively and negatively supercoiled DNA minicircles carrying a single protein binding site. Mobility shifts of complexes relative to naked DNAs appeared to be a result of i) the unwinding; of ii) an increase in the molecular frictional coefficient, which led to a retardation; of iii) bending, in the particular case of CAP, which induced an acceleration; and of iv) looping, in the case of lac repressor, which also resulted in an acceleration. Under conditions where the migration of the naked topoisomers was V-like (topoisomer mobility showed the same linear increase with both negative and positive supercoilings; Zivanovic et al. (1986) J. Mol. Biol., 192, 645-660), the protein unwinding contribution to mobility was assumed to be identical to that experimentally observed in the case of a thermal unwinding: all negatively supercoiled topoisomers were retarded and all positively supercoiled topoisomers were accelerated to the same extent. In contrast, the mobility contribution of the frictional term, as well as those of bending and looping, appeared to vary strongly with the magnitude of the supercoiling, but only weakly with its polarity. As a consequence, these latter contributions may approximately cancel when one is measuring the difference between the shifts observed for two comigrating, negatively and positively supercoiled, topoisomers, allowing the unwinding to be calculated. While estimates obtained for EcoR I, 23 +/- 3 degrees, and CAP, about 29 degrees, were in good agreement with previous measurements using topoisomerase I, the value found for lac repressor, 13 to 16 degrees, was significantly smaller.     

Synthetic lac operator mediates repression through lac repressor when introduced upstream and downstream from lac promoter.

Plasmids were constructed which carry a synthetic lac operator at various distances from the lac promoter. They were tested in vivo for function in the presence and absence of lac repressor. We found significant repression when the lac operator is situated at the 3' end of the lac I gene or at the 5' end of the lac Z gene. When lac operators are inserted at both sites, we found a greater than 150-fold repression. The complex between lac repressor and DNA carrying these two lac operators is exceedingly stable in vitro suggesting that one tetrameric lac repressor may bind to both lac operators.     

Role of HU and DNA supercoiling in transcription repression: specialized nucleoprotein repression complex at gal promoters in Escherichia coli

Efficient repression of the two promoters P1 and P2 of the gal operon requires the formation of a DNA loop encompassing the promoters. In vitro, DNA looping-mediated repression involves binding of the Gal repressor (GalR) to two gal operators (OE and OI) and binding of the histone-like protein HU to a specific locus (hbs) about the midpoint between OE and OI, and supercoiled DNA. Without DNA looping, GalR binding to OE partially represses P1 and stimulates P2. We investigated the requirement for DNA supercoiling and HU in repression of the gal promoters in vivo in strains containing a fusion of a reporter gene, gusA or lacZ, to each promoter individually. While the P1 promoter was found to be repressible in the absence of DNA supercoiling and HU, the repression of P2 was entirely dependent upon DNA supercoiling in vivo. The P2 promoter was fully derepressed when supercoiling was inhibited by the addition of coumermycin in cells. P2, but not P1, was also totally derepressed by the absence of HU or the OI operator. From these results, we propose that the repression of the gal promoters in vivo is mediated by the formation of a higher order DNA-multiprotein complex containing GalR, HU and supercoiled DNA. In the absence of this complex, P1 but not P2 is still repressed by GalR binding to OE. The specific nucleoprotein complexes involving histone-like proteins, which repress promoter activity while remaining sensitive to inducing signals, as discussed, may occur more generally in bacterial nucleoids.     

Wrapping of flanking non-operator DNA in lac repressor-operator complexes: implications for DNA looping

In our studies of lac repressor tetramer (T)-lac operator (O) interactions, we observed that the presence of extended regions of non-operator DNA flanking a single lac operator sequence embedded in plasmid DNA produced large and unusual cooperative and anticooperative effects on binding constants (Kobs) and their salt concentration dependences for the formation of 1:1 (TO) and especially 1:2 (TO2) complexes. To explore the origin of this striking behavior we report and analyze binding data on 1:1 (TO) and 1:2 (TO2) complexes between repressor and a single O(sym) operator embedded in 40 bp, 101 bp, and 2514 bp DNA, over very wide ranges of [salt]. We find large interrelated effects of flanking DNA length and [salt] on binding constants (K(TO)obs, K(TO2)obs) and on their [salt]-derivatives, and quantify these effects in terms of the free energy contributions of two wrapping modes, designated local and global. Both local and global wrapping of flanking DNA occur to an increasing extent as [salt] decreases. Global wrapping of plasmid-length DNA is extraordinarily dependent on [salt]. We propose that global wrapping is driven at low salt concentration by the polyelectrolyte effect, and involves a very large number (>/similar 20) of coulombic interactions between DNA phosphates and positively charged groups on lac repressor. Coulombic interactions in the global wrap must involve both the core and the second DNA-binding domain of lac repressor, and result in a complex which is looped by DNA wrapping. The non-coulombic contribution to the free energy of global wrapping is highly unfavorable ( approximately +30-50 kcal mol(-1)), which presumably results from a significant extent of DNA distortion and/or entropic constraints. We propose a structural model for global wrapping, and consider its implications for looping of intervening non-operator DNA in forming a complex between a tetrameric repressor (LacI) and one multi-operator DNA molecule in vivo and in vitro. The existence of DNA wrapping in LacI-DNA interactions motivates the proposal that most if not all DNA binding proteins may have evolved the capability to wrap and thereby organize flanking regions of DNA.     

Effect of DNA looping on the induction kinetics of the lac operon

The induction of the lac operon follows cooperative kinetics. The first mechanistic model of these kinetics is the de facto standard in the modeling literature [Yagil, G., Yagil, E., 1971. On the relation between effector concentration and the rate of induced enzyme synthesis. Biophys. J. 11, 11-17]. Yet, subsequent studies have shown that the model is based on incorrect assumptions. Specifically, the repressor is a tetramer with four (not two) inducer-binding sites, and the operon contains two auxiliary operators (in addition to the main operator). Furthermore, these structural features are crucial for the formation of DNA loops, the key determinants of lac repression and induction. Indeed, the repression is determined almost entirely (>95%) by the looped complexes [Oehler, S., Eismann, E.R., Kr?mer, H., Müller-Hill, B., 1990. The three operators of the lac operon cooperate in repression. EMBO J. 9(4), 973-979], and the pronounced cooperativity of the induction curve hinges upon the existence of the looped complexes [Oehler, S., Alberti, S., Müller-Hill, B., 2006. Induction of the lac promoter in the absence of DNA loops and the stoichiometry of induction. Nucleic Acids Res. 34(2), 606-612]. Here, we formulate a model of lac induction taking due account of the tetrameric structure of the repressor and the existence of looped complexes. We show that: (1) The kinetics are significantly more cooperative than those predicted by the Yagil and Yagil model. The cooperativity is higher because the formation of looped complexes is easily abolished by repressor-inducer binding. (2) The model provides good fits to the repression data for cells containing wild-type tetrameric or mutant dimeric repressor, as well as the induction curves for 6 different strains of Escherichia coli. It also implies that the ratios of certain looped and non-looped complexes are independent of inducer and repressor levels, a conclusion that can be rigorously tested by gel electrophoresis. (3) Repressor overexpression dramatically increases the cooperativity of the induction curve. This suggests that repressor overexpression can induce bistability in systems, such as growth of E. coli on lactose, that are otherwise monostable.     

Recombination by resolvase is inhibited by lac repressor simultaneously binding operators between res sites

The Tn3 resolvase requires that the two recombination (res) sites be aligned as direct repeats on the same molecule for efficient recombination to occur. To test whether resolvase must contact the DNA between res sites as predicted by tracking models, we have determined the sensitivity of recombination to protein diffusion blockades. Recombination between two res sites is unaffected either by lac repressor or bacteriophage T7 RNA polymerase being bound between them. Yet recombination is inhibited by lac repressor if the res site is bounded by a lac operator on both sides. We demonstrate that lac repressor will bind to more than one DNA site under the conditions used to assay recombination. This result suggests that lac repressor can inhibit resolvase by forming a DNA loop that isolates a res site topologically. These results do not support a tracking model for resolvase but suggest that the structure and topology of the DNA substrate is important in the formation of a synapse between res sites.     

Specific binding of lac repressor to linear versus circular polyoperator molecules

Gel shift assays were used to examine the binding of the lactose (lac) repressor to polyoperator DNA molecules. Specific binding was differentiated from nonspecific DNA association by (i) equilibrating repressor-operator complexes below the nonspecific association constant and (ii) demonstrating the effects of the inducer isopropyl beta-D-thiogalactoside (IPTG) on the formation of repressor-operator complexes. With the linear polyoperator molecules, all eight operator sites could be simultaneously bound by distinct repressors. However, with circular molecules, the eight operator sites were saturable by repressor only in the nicked circular state and not in the covalently closed circular form. Under the experimental conditions used, there was no evidence of bifunctional repressor binding or loop formation. The results suggest that the conformational perturbation of DNA that occurs upon specific repressor binding was retained in topologically closed molecules and could modify other operator sites so as to make them unavailable for specific binding.     

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.