Interaction of tight binding repressors with lac operators. An analysis by DNA-footprinting

To increase our understanding of protein-DNA interaction in general, and in particular that of lac repressor with lac operator, we have investigated the interaction of tight binding (Itb) repressors with wild type (WT) operator and Oc operators. Nine Oc and a WT operator were cloned and sequenced. Three different Oc and an O+ were then chosen for the footprint analysis of six Itb repressors and WT repressor. Distinct protection patterns for the various repressor-operator pairs were observed at low repressor concentrations whereas, at high repressor concentrations, a stretch of 24 bases of the lower strand of the four different operators was protected in most cases. This protection pattern at high repressor concentration was almost completely redundant for all repressor-operator pairs, in spite of the fact that the affinities of the various pairs differed by more than three orders of magnitude. Two exceptions to this general observation were the two tight binding repressors R67 and R78a. These had been mapped in a region that codes for amino acid residues involved in subunit interaction. The two repressors showed reduced protection of O+ and of some Oc operators at the 3' (right) end of the lower strand. Dimethylsulfoxide, which is known to increase the affinity of O+ for repressor, did not increase the number of bases protected by WT repressor on the lower strand of O+. The footprinting results presented here clearly demonstrate that lac repressor can maximally protect about 24 bases of the lower strand of the operator and that the number and kind of interactions occurring in this region determine the strength of the repressor-operator interaction.     

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.     

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.     

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.     

The interaction of the recognition helix of lac repressor with lac operator.

We have constructed a system which allows systematic testing of repressor--operator interactions. The system consists of two plasmids. One of them carries a lac operon in which lac operator has been replaced by a unique restriction site into which synthetic operators can be cloned. The other plasmid carries the gene coding for the repressor, in our case a semisynthetic lacI gene of which parts can be exchanged in a cassette-like manner. A galE host allows us to select for mutants which express repressors with altered specificities. Here we report the change of specificity in the lac system by changing residues 1 and 2 of the recognition helix of lac repressor. The specificity changes are brought about cooperatively by the change of both residues. Exchanges of just one residue broaden the specificity. Our results hint that the recognition helix of lac repressor may possibly have the opposite orientation to those in Lambda cro protein or 434 CI repressor.     

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.     

Mutations partially inactivating the lactose repressor of Escherichia coli

After treatment with N-methyl-N'-nitro-N-nitrosoguanidine, 133 independent mutants of a haploid strain of Escherichia coli able to use phenyl-beta-galactoside as a carbon source were obtained. The galactoside was specific in selecting for mutants with increases in their uninduced levels of beta-galactosidase. Virtually all mutants (37 in a subsample of 38) carried mutations in the lac repressor gene. There were two classes of repressor mutants. As well as the commonly identified class of mutants with completely inactivated repressors, there was a frequent class of mutants (21/37) whose repressors were partially inactivated. Most of these (15/21) repressed beta-galactosidase synthesis 4 to 50 times less than wild type, but were more numerous in the lower part of this range. Their beta-galactosidase was inducible to levels characteristic of the parent strain. The repressor activities were diverse and stably expressed under routine growth conditions. The decreased activity did not result from the formation of temperature-sensitive repressors. None of the mutants with completely inactivated repressors appeared to carry UAG or UGA chain-terminating codons. On the assumption that the partially defective repressor mutants carried missense mutations, it is argued that missense mutations in the lac repressor gene modify the repressor's affinity for the operator with high probability. An explanation is proposed for the apparent sensitivity of this repressor function to partial inactivation as the result of amino acid substitutions.     

P22 repressor mutants deficient in co-operative binding and DNA loop formation.

We show here, both in vivo and in vitro, that P22 repressor binds co-operatively to operator sites separated by an integral number of turns of the DNA helix. We measure this co-operativity in vivo using an assay in which repression of a promoter requires co-operative binding of P22 repressors to two separated (non-adjacent) operator sites. We report the isolation of mutant repressors that have high affinity for single operator sites, but are defective in co-operative binding. Six different mutants, all bearing single amino acid changes in the carboxyl domain, have been isolated. We purified the two mutants most deficient in co-operative binding, and found that they bind non-co-operatively in vitro to adjacent as well as to non-adjacent pairs of operator sites.     

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.     

lac Operator nucleosomes. 2. lac Nucleosomes can change conformation to strengthen binding by lac repressor

We have shown previously that lac repressor binds specifically and quantitatively to lac operator restriction fragments which have been complexed with histones to form artificial nucleosomes (203 base pair restriction fragment) or core particles (144 base pair restriction fragment. We describe here a quantitative method for determining the equilibrium binding affinities of repressor for these lac reconstitutes. Quantitative analysis shows that the operator-histone reconstitutes may be grouped into two affinity classes: those with an affinity for repressor close to that of naked DNA and those with an affinity 2 or more orders of magnitude less than that of naked DNA. All particles in the lac nucleosome preparations bind repressor with high affinity, but the lac core particle preparations contain particles of both high and low affinities for repressor. Formaldehyde cross-linking causes all high-affinity species to suffer a 100-fold decrease in binding affinity. In contrast, there is no effect of cross-linking on species of low affinity. Therefore, the ability of a particle to be bound tightly by repressor depends on a property of the particle which is eliminated by cross-linking. Control experiments have shown that chemical damage to the operator does not accompany cross-linking. Therefore, the property sensitive to cross-linking must be the ability of the particle to change conformation. We infer that the particles of low native affinity, like cross-linked particles, are of low affinity because of an inability to facilitate repressor binding by means of this conformational change. Dimethyl suberimidate cross-linking experiments show that histone-histone cross-linking is sufficient to preclude high-affinity binding. Thus, the necessary conformational change involves a nucleosome histone core event. We find that the ability of a particle to undergo a repressor-induced facilitating conformational change appears to depend on the position of the operator along the DNA binding path of the nucleosome core. We present a general model which proposes that nucleosomes are divided into domains which function differentially to initiate conformational changes in response to physiological stimuli.     

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.     

Degradation of the DNA-binding domain of wild-type and i-d lac repressors in Escherichia coli.

It has been shown that 28 transdominant mutant lac repressors which have lost operator DNA-binding ability in vivo and in vitro, but still bind inducer and are able to form tetramers (i-d repressors), could be divided into two groups by their capacity or incapacity to bind non-specifically to the phosphate groups of the DNA backbone. All but one of 15 analysed i-d repressors with amino acid substitutions to the C-terminal of residue 52 showed uneffected non-specific DNA binding. All 13 tested i-d repressors with amino acid substitutions to the N-terminal of residue 53 did not bind to double-stranded DNA, and 11 of these repressors derived from missense mutations in the lacI gene were endogenously degraded. The degradation in vivo only affects the amino-terminal 50-60 residues producing a mutant-specific pattern of stable repressor fragments. These fragments are tetrameric and capable of binding inducer in vivo and in vitro. The proteolytic attack presumably takes place during synthesis of the i-d repressors, since the resulting fragments are stable, both in vivo (as shown by a pulse-chase experiment) and in vitro. The proteolysis in vivo depends on the growth conditions of the bacteria and is higher in cells grown in minimal media than in rich media. Wild-type repressor is only susceptible to limited proteolysis in cells grown in minimal media but not in cells grown in rich media. The results suggest that the majority of the sequence alterations before residue 53 in missense mutant i-d lac repressor proteins affect the three-dimensional structure of the amino-terminal DNA-binding domain of the repressor protein, making it susceptible to proteolytic attack by one or several intracellular proteases.     

Interaction of the tight-binding I12-X86 lac repressor with non operator DNA: salt dependence of complex formation.

The interaction of the wild-type lac repressor and its tight binding double mutant I12-X86 with a non operator-210 base pair-DNA fragment has been investigated using the nitrocellulose filter binding assay. While the affinity of the double mutant for this non specific DNA is increased as compared to that of the wild-type repressor, the number of ions released from the vicinity of the DNA upon complex formation is less important for the mutant than for the wild-type. These results demonstrate that the adaptation in the recognition surface of the repressor recently proposed by Mossing et al (J. Mol. Biol., 1985, 186, 295-305) in the case of an Oc mutant may be a more general phenomenon.     

The lac repressor

Few proteins have had such a strong impact on a field as the lac repressor has had in Molecular Biology. Over 40 years ago, Jacob and Monod [Genetic regulatory mechanisms in the synthesis of proteins, J. Mol. Biol. 3 (1961) 318] proposed a model for gene regulation, which survives essentially unchanged in contemporary textbooks. It is a cogent depiction of how a set of 'structural' genes may be coordinately transcribed in response to environmental conditions and regulates metabolic events in the cell. In bacteria, the genes required for lactose utilization are negatively regulated when a repressor molecule binds to an upstream cis activated operator. The repressor and its operator together form a genetic switch, the lac operon. The switch functions when inducer molecules alter the conformation of the repressor in a specific manner. In the presence of a particular metabolite, the repressor undergoes a conformational change that reduces its affinity for the operator. The structures of the lac repressor and its complexes with operator DNA and effector molecules have provided a physical platform for visualizing at the molecular level the different conformations the repressor and the molecular basis for the switch. The structures of lac repressor, bound to its operator and inducer, have also been invaluable for interpreting a plethora of biochemical and genetic data.     

Use of a ligand-screening procedure to study the interaction of S. cerevisiae alpha 2 repressor with its operator sequence.

A simple and rapid screening procedure was developed to study the interaction of the S. cerevisiae alpha 2 repressor with its operator sequence. An E. coli expression vector was constructed in which the alpha 2 coding sequence was placed under control of the lac promoter. Bacterial colonies containing this vector could be lysed and assayed directly for binding of wild-type and mutant operator sequences when grown on nitrocellulose filters. alpha 2 assayed in this way showed the same sequence specificity as determined in vivo. Pools of mutant alpha 2 repressors in which the codons for Arg185 or Ser181 in the homeodomain region were randomized were created by cassette mutagenesis. These pools of mutants were screened with the wild-type operator sequence to determine allowed amino acid substitutions at each position. Results suggest that both Arg185 and Ser181 have a role in high affinity operator binding.     

Lac repressor purification without inactivation of DNA binding activity.

A procedure has been developed which eliminates the commonly observed inactivation of the DNA binding activity of the lac repressor during purification. The operator binding activity of the repressor obtained by this method is 100 +/- 10%. The repressor can be stored frozen indefinitely without losing its affinity for DNA.     

Studies on gene control regions. VI. The 5- methyl of thymine, a lac repressor recognition site.

Three site specific deoxyuridine analogs of lac operator were tested for binding with wild type (SQ) and tight binding (QX86) lac repressors. Insertion of uracil for thymine at site 13 (our nomenclature) significantly reduced the dissociation half-life of QX86 repressor for lac operator DNA (21 vs 1.2 min). Two other sites (6 and 7) are affected to a much lesser extent.     

Lambda repressor mutations that increase the affinity and specificity of operator binding

Intragenic, second-site reversion has been used to identify amino acid substitutions that increase the affinity and specificity of the binding of lambda repressor to its operator sites. Purified repressors bearing the second-site substitutions bind operator DNA from 3 to 600 fold more strongly than wild type; these affinity changes result from both increased rates of operator association and decreased rates of operator dissociation. Three of the revertant substitutions occur in the alpha 2 and alpha 3 DNA binding helices of repressor and seem to increase affinity by introducing new salt-bridges or hydrogen bonds with the sugar-phosphate backbone of the operator site. The fourth substitution alters the alpha 5 dimerization helix of repressor and appears to increase operator affinity indirectly.