Tight-binding repressors of the lac operon: selection system and in vitro analysis.

The isolation and characterization of altered repressors of the lac operon which have an increased affinity for an operator should give useful clues about the molecular basis for the very tight and specific interaction between repressor and operator. A selection system has been devised which allows the isolation of such repressor mutants. This system selects for mutant repressors which can overcome lac operator-constitutive (Oc) mutations. By using in vivo assays, 24 candidates were obtained which, compared with wild type, have an increased trans effect of their repressor on one or several Oc operators. Three of these candidates have been investigated in vitro; the affinity of their repressor for inducer was unchanged, whereas the affinity for wild-type operator was increased 15-, 86-, and 262-fold, respectively.     

Mutant lac repressors with new specificities hint at rules for protein--DNA recognition.

Proteins which recognize specific sequences of DNA play a fundamental role in the regulation of protein synthesis in all organisms. A particular helix of the bacterial protein lac repressor recognizes the bases in the major groove of the lac operator. We show that the first two residues of this recognition helix interact independently with two base pairs. This allows us in many cases to predict repression as an indicator of strength of the repressor-operator complex. Rules of recognition can be derived for 16 symmetric operators. They also apply to the gal repressor and possibly to other bacterial repressors.     

Affinities of tight-binding lactose repressors for wild-type and pseudo-operators

Five tight-binding (Itb) mutants of the Escherichia coli lactose (lac) repressor have been characterized with regard to their non-specific affinity for DNA and their specific affinity for the wild-type operator and several sequence-altered (pseudo-) operators. Repressor-operator association rates were determined in the presence or absence of competitor DNA, dissociation rates of repressor from various DNA fragments were measured, and equilibrium competition for repressor binding was examined for several pseudo-operator DNAs. The mutant repressors exhibited increased non-specific affinity for DNA, and variable increases in affinity for sequence-altered operators. The known positions of amino acid substitutions for three of these Itb repressors support suggestions that residues 51 to 64 are important for operator recognition in addition to residues 1 to 50.     

Effects of dominant-negative lac repressor mutations on operator specificity and protein stability

The specific binding of dominant-negative (I-d) lactose (lac) repressors to wild-type (wt) as well as mutant (Oc) lac operators has been examined to explore the sequence-specific interaction of the lac repressor with its target. Mutant lacI genes encoding substitutions in the N-terminal 60 amino acids (aa) were cloned in a derivative of plasmid pBR322. Twelve of these lacI-d missense mutations were transferred from F'lac episomes using general genetic recombination and molecular cloning, and nine lacI missense mutations were recloned from M13-lacI phages [Mott et al., Nucl. Acids Res. 12 (1984) 4139-4152]. The mutant repressors were examined for polypeptide size and stability, for binding the inducer isopropyl-beta-D-thiogalactoside (IPTG), as well as binding to wt operator. The mutant repressors' affinities for wt operator ranged from undetectable to about 1% that of wt repressor, and the mutant repressors varied in transdominance against repressor expressed from a chromosomal lacIq gene. Six of the I-d repressors were partially degraded in vivo. All repressors bound IPTG with approximately the affinity of wt repressor. Repressors having significant affinity for wt operator or with substitutions in the presumed operator recognition helix (aa 17-25) were examined in vivo for their affinities for a series of single site Oc operators. Whereas the Gly-18-, Ser-18- and Leu-18-substituted repressors showed altered specificity for position 7 of the operator [Ebright, Proc. Natl. Acad. Sci. USA 83 (1986) 303-307], the His-18 repressor did not affect specificity. This result may be related to the greater side-chain length of histidine compared to the other amino acid substitutions.     

Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator 01 through alternative conformations of its DNA-binding domain

The lac repressor-operator system is a model system for understanding protein-DNA interactions and allosteric mechanisms in gene regulation. Despite the wealth of biochemical data provided by extensive mutations of both repressor and operator, the specific recognition mechanism of the natural lac operators by lac repressor has remained elusive. Here we present the first high-resolution structure of a dimer of the DNA-binding domain of lac repressor bound to its natural operator 01. The global positioning of the dimer on the operator is dramatically asymmetric, which results in a different pattern of specific contacts between the two sites. Specific recognition is accomplished by a combination of elongation and twist by 48 degrees of the right lac subunit relative to the left one, significant rearrangement of many side chains as well as sequence-dependent deformability of the DNA. The set of recognition mechanisms involved in the lac repressor-operator system is unique among other protein-DNA complexes and presents a nice example of the adaptability that both proteins and DNA exhibit in the context of their mutual interaction.     

Non-contacted bases affect the affinity of synthetic P22 operators for P22 repressor.

The affinity of synthetic P22 operators for P22 repressor varies with the base sequence at the operator's center. At 100 mM KCl, the affinity of these operators for P22 repressor varies over a 10-fold range. Dimethylsulfate protection experiments indicate that the central bases of the P22 operator are not contacted by the repressor. The KD for the complex of P22 repressor with an operator bearing central T-A bases (9T) increases less than 2-fold between 50 and 200 mM KCl, whereas the KD for the complex of repressor with an operator bearing central C-G bases (9C) increases 10-fold in the same salt range. The DNase I cleavage patterns of both bound and unbound P22 operators also vary with central base sequence. The DNase I pattern of the repressor-9C operator complex changes markedly with salt concentration, whereas that of the 9T operator-repressor complex does not. These changes in nuclease digestion pattern thereby mirror the salt-dependent changes in the P22 operator's affinity for repressor. P22 repressor protects the central base pair of the 9T operator from cleavage by the intercalative cleavage reagent Cu(I)-phenanthroline, while repressor does not protect the central bases of the 9C operator. Together these data indicate that central base pairs affect P22 operator strength by altering the structure of the unbound operator and the repressor-operator complex.     

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.     

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.     

Physical properties of DNA in vivo as probed by the length dependence of the lac operator looping process

Plasmid constructs containing a wild-type (O+) lac operator upstream of an operator-constitutive (Oc) lac control element exhibit a length-dependent, oscillatory pattern of repression of expression of the regulated gene as interoperator spacing is varied from 115 to 177 base pairs (bp). Both the length dependence and the periodicity of repression are consistent with a thermodynamic model involving a stable looped complex in which bidentate lac repressor interacts simultaneously with both O+ and Oc operators. The oscillatory pattern of repression with distance occurs with a period approximating the helical repeat of DNA and presumably reflects the necessity for proper alignment of interacting operators along the helical face of the DNA. In the length regime examined, the presence of the upstream operator enhances repression between 6-fold and 50-fold depending upon phasing. This reflects a torsional rigidity of DNA in vivo that is consistent with in vitro measurements. The oscillatory pattern of repression is best fit with a period of either 9.0 or 11.7 bp/cycle but not 10.5 bp/cycle. This periodicity is interpreted as reflecting the average helical repeat of the 40-bp interoperator region of plasmid DNA in vivo, suggesting that the local helical repeat of DNA in vivo may differ significantly from 10.5 bp/turn. The apparent persistence length needed to fit the data (aapp) is only one-fifth the standard in vitro value. This low value of aapp may be due in part to DNA bending induced by catabolite activator protein (CAP) bound to its site between the interacting operators.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence-specific DNA-protein interaction: the lac represser

A model is proposed for lac repressor-lac operator binding which accounts for the tetrameric subunit structure of the lac repressor and for factors involved in the strength, specificity and regulation of repressor-operator interaction. The model employs a π-helix in the amino terminal 25 residues of the lac repressor whereby three tyrosine residues of each subunit intercalate between base pairs of the lac operator. For the outer palindromic sequences of the operator, base specificity is provided by amino acids adjacent to the carboxyl sides of the tyrosine residues of two of the subunits. The inner palindromic sequences which bind the other two subunits form stems of hairpin loops in the operator. Base specificity for these two subunits is provided by amino acids adjacent to the amino sides of the tyrosine residues. In addition to 12 intercalated tyrosine residues, the model provides for a total of at least eight electrostatic interactions and ten sequence-specific hydrogen bonds.     

Binding of polynucleotides to fibroblasts. Effects of complex formation with vinyl analogs of nucleic acids

The nitrocellulose filter assay was used to study the effect of the DNA denaturants glycerol and dimethylsulfoxide (Me2SO) on the lac repressor-operator interaction. Both glycerol and Me2SO decrease the rate of dissociation (kb) of the repressor-operator complex but do not significantly alter the rate of association of repressor and operator. In the presence of 10% Me2SO an almost 10-fold increase of affinity of repressor for operator is observed. A small increase in affinity of repressor for Escherichia coli DNA, chicken blood DNA, and poly(dA-dT) is also found. The results lead to the conclusion that lac repressor when interacting with the operator causes local destabilization of the DNA.     

lac repressor mutants with double or triple exchanges in the recognition helix bind specifically to lac operator variants with multiple exchanges.

Several lac repressor mutants have been isolated which repress beta-galactosidase synthesis in Escherichia coli up to 200-fold. They do so by binding specifically to particular symmetrical lac Oc operator variants. The mutations in the lac repressor are localized in two separate parts of the recognition helix comprising (i) residues 1 and 2 which interact with base pairs 4 and 5 of lac operator and (ii) residue 6 which recognizes operator base pair 6. Mutations of residues 1 and 2 may be combined with a mutation of residue 6. The resulting mutant protein binds specifically to an operator variant with three symmetric exchanges in base pairs 4, 5 and 6.     

Methylphosphonates as probes of protein-nucleic acid interactions.

Deoxydinucleoside methylphosphonates were prepared by chemical synthesis and were introduced stereospecifically into the lac operator at two sites. These sites within d(ApApTpTpGpTpGpApGpCpGpGpApTpApApCpApApTpT), segment I, and d(ApApTpTpGpTpTpApTpCpCpGpCpTpCpApCpApApTpT), segment II, are indicated by p. Each segment containing a chiral methylphosphonate was annealed to the complementary unmodified segment. The interactions of these four modified lac operators with lac repressor were analyzed by the nitrocellulose filter binding assay. Introduction of either chiral phosphonate in segment II had little effect on the stability of the repressor-operator complex. When methylphosphonates were introduced into segment I, the affinity of lac repressor for the modified operators was shown to be dependent on the stereochemical configuration of the methylphosphonate.     

Interaction of effecting ligands with lac repressor and repressor-operator complex.

The equilibrium association constants for the binding of a wide variety of effecting ligands of the lac repressor were measured by equilibrium dialysis. Also, detailed investigations of the apparent rate of dissociation of repressor-operator comples as a function of ligand concentration were carried out for several inducers and anti-inducers. The affinity of repressor-ligand comples for operator DNA was evaluated from the specific rate constants at saturating concentrations of effecting ligand. By fitting the experimental data depicting the functional dependence of the rate of dissociation upon ligand concentrations to calculated curves, assuming simple models of the induction mechanism, the equilibrium association constant for the binding of effecting ligand to repressor-operator comples was determined. Inducers reduce the affinity of lac repressor for operator DNA by a factor of approximately 1000 under standard conditions; the extent of destabilization depends on Mg2+ ion concentration. Anti-inducers increase the affinity of repressor for operator at most a factor of five. Only one neutral ligand, which binds to repressor without altering the stability of repressor-operator comples, was found. No homotropic or heterotropic interactions in the binding of effecting ligands either to repressor or to repressor-operator complex are evident.     

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.     

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 stable loops in vitro with supercoiled wild-type lac DNA containing all three natural lac operators

We have analyzed protein-DNA complexes formed between lac repressor and linear or differently supercoiled lac DNA (802 or 816 base-pairs in length), which carry all three natural lac operators (O1, O2 and O3) in their wild-type sequence context and spacing and compared them with constructs that contain specifically mutated "pseudo-operators" O2 or O3. We used gel retardation assays to identify the nature of the complexes according to their characteristic electrophoretic mobility and dissociation rate measurements to determine their stability. With linear DNA we found only indirect evidence for loop formation between O1 and O2. In covalently closed DNA minicircles the formation of a loop between O1 and O2 could be demonstrated by the observation that O1-O2 containing DNA with low negative supercoiling (sigma = -0.013 and less) is constricted by binding of lac repressor, resulting in an increased electrophoretic mobility. At elevated negative supercoiling (sigma = -0.025, -0.037, -0.05) O1-O2 containing DNA complexed with lac repressor migrates significantly slower than the corresponding O1-DNA, indicating loop formation. The dissociation of lac repressor-operator complexes is decreased with increasing negative supercoiling for all tested operator combinations of O1, O2 and O3. However, in the presence of at least two natural lac operators on the same DNA minicircle the enhancement of stability is particularly large. This indicates that a DNA loop is formed between these two lac operators, O1 and O2 as well as O1 and O3, since negative supercoiling is known specifically to promote the formation of looped structures. Additionally, we observe a dependence of dissociation rate on the spatial alignment of the operators as a result of changing helical periodicity in differently supercoiled DNA and consider this to be further evidence for loop formation between O1 and O2 as well as O1 and O3.