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At least 19 recordsLinked to original sources
PubMed1981

Lac repressor - lac operator interaction. Circular dichroism study.

The interaction between lac repressor and a small operator DNA fragment have been examined by circular dichroism spectroscopy. The binding of lac repressor on the operator induces a conformation change of the DNA which is different from that observed upon non specific binding on non operator DNA. The CD titration curve indicates that the stoechiometry of interaction is complex. A two operators-one repressor complex was found. This result was confirmed by a gel filtration experiment.

Base Sequence↗
PubMed1992

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.

Bacteriophage lambda↗
PubMed1982

lac repressor-lac operator interaction: NMR observations.

We show here the changes in the NMR spectra of the Escherichia coli lac repressor when bound to isolated lac operator DNA. The observations focus on the aromatic residues--four tyrosines and a single histidine--in the amino-terminal DNA binding domain of the lac repressor. There is a good correlation between chemical shift changes seen by 19F NMR when compared with 1 H NMR of otherwise identical repressor--DNA complexes. The results suggest that the tyrosines do not intercalate in the DNA. The NMR spectral changes with similarly sized DNA fragments, not containing the lac operator DNA sequence, are different. Thus, the amino-terminal domain of the lac repressor is independently capable of discriminating between lac operator and nonspecific DNA sequences. There can be two amino-terminal fragments per operator in the specific complex.

Amino Acid Sequence↗
PubMed1996

Compensating effects of opposing changes in putrescine (2+) and K+ concentrations on lac repressor-lac operator binding: in vitro thermodynamic analysis and in vivo relevance.

Ion concentrations (K+, Glu-) in the cytoplasm of growing Escherichia coli cells increase strongly with increases in the osmolarity of a defined growth medium. While in vitro experiments demonstrate that the extent of protein-nucleic acid interactions (PNAI) depends critically on salt concentration, in vivo measurements indicate that cells maintain a relatively constant extent of PNAI independent of the osmolarity of growth. How do cells buffer PNAI against changes in the cytoplasmic environment? At high osmolarity, the increase in macromolecular crowding which accompanies the reduction in amount of cytoplasmic water in growing cells appears quantitatively sufficient to compensate for the increase in [K+]. At low osmolarity, however, changes in crowding appear to be insufficient to compensate for changes in [K+], and additional mechanisms must be involved. Here we report quantitative determinations of in vivo total concentrations of polyamines (putrescine(2+), spermidine(3+)) as a function of osmolarity (OsM) of growth, and in vitro binding data on the effects of putrescine concentration on a specific PNAI (lac repressor-lac operator) as a function of [K+]. The total concentration of putrescine in cytoplasmic water decreases at least eightfold from low osmolarity (approximately 64 mmol (l H2O)-1 at 0.03 OsM) to high osmolarity (approximately 8 mmol (l H2O)-1 at 1.02 OsM). Over this osmotic range the total [K+] increases from approximately 0.2 mol (l H2O)-1 to approximately 0.8 mol (lH2O)-1. We find that the effect of putrescine concentration on the repressor-operator interaction in vitro is purely competitive and is quantitatively described by a simple competition formalism in which lac repressor behaves a a specific-binding oligocation (ZR = 8+/-3). We demonstrate that this thermodynamic result is consistent with a structural analysis of the number of positively charged side-chains on two DNA binding domains of repressor which interact with the phosphodiester backbone of the operator site. Since this oligocation character of the binding surface of DNA-binding proteins appears to be general, we propose the competitive effects of putrescine and K+ concentrations on the strength of specific binding are general. At low osmolarity, compensating changes in putrescine and K+ concentration in response to changes in external osmolarity provide a general mechanism for E. coli to vary cytoplasmic osmolarity while maintaining a constant extent of PNAI.

Base Sequence↗
PubMed1993

Origin of the asymmetrical contact between lac repressor and lac operator DNA.

The Escherichia coli lac operator DNA contains two sequence repeats related by a pseudo-dyad axis. Deviations from symmetry, in the central 21 bp sequence, occur at two pairs of symmetrically related sites (+15/+7, +13/+9) and at the central base-pair +11. Mutational analysis and DNA protection studies have suggested asymmetric interactions of lac repressor along this sequence. Previous biophysical studies on the lac repressor-operator system have typically employed symmetrized operator sequences to simplify analysis. As a result, it has remained difficult to assess the importance of the naturally occurring sequence deviations from symmetry. Here, 19F-NMR is used to determine if the wild-type E. coli lac operator DNA sequence itself specifies a pair of distinct half-site interactions with lac repressor DNA binding domains. To observe protein interactions simultaneously at operator half-sites using 19F-NMR, three pairs of naturally occurring, symmetry related thymine residues (at +6/+16, +8/+14 and +1/+21) were substituted pair-wise by 5-fluorodeoxyuridines (5-FdU). Two polypeptides corresponding to the N-terminal DNA binding domain of lac repressor "headpiece", residues 1 to 56 and 1 to 64, were employed to remove the steric constraints of subunit interaction in the wild-type tetramer. Spectral changes associated with headpiece binding to left side DNA sequences differ from those caused by binding to equivalent sequences on the right half-site. These results are similar to non-symmetric intact tetramer repressor interactions specified by the DNA sequence. Three mutant lac operator sequences with increased symmetry, bearing FdU substitutions were used to identify the relative importance of the three naturally asymmetric positions. Symmetrizing one pair of these sites alone or in addition to removing the central base-pair failed to produce identical NMR signal changes characteristic of symmetric headpiece-DNA complexes. However, symmetrizing both asymmetric pairs gave chemical shift changes expected from symmetric protein-DNA complexes. We propose that key interactions with the left side +9 (G.C) are altered at the symmetrically related right side +13 (A.T). The data show that the DNA sequence at +13 influences interactions three base-pairs away.

Base Sequence↗
PubMed2001

Radiosensitivity of DNA in a specific protein-DNA complex: the lac repressor-lac operator complex.

PURPOSE: To calculate the probability of radiation-induced frank strand breakage (FSB) at each nucleotide in the Escherichia coli lac repressor-lac operator system using a simulation procedure. To compare calculated and experimental results. To asses the contribution of DNA conformational changes and of the masking by the protein to DNA protection by the repressor. MATERIALS AND METHODS: Two structures of the complex were extracted from the PDB databank: crystallography- and NMR-based structures. Calculations were made of the accessibility of the atoms mainly involved in strand breakage (H4' and H5') to O&Hdot; and of the FSB probabilities, along: (1) DNA in the complex; (2) DNA in the complex depleted of the repressor; and (3) a linear DNA having the same sequence. An 80bp fragment bearing the operator was irradiated alone or in presence of the repressor. The relative probabilities of FSB at each nucleotide were determined using sequencing gel electrophoresis. RESULTS: Calculations predict modulation of the accessibility of H4' and H5' atoms and of the probabilities of FSB along the DNA fragments of complexes. This is due to the protein-induced conformational change and to masking by bound protein. The best agreement with the experimental FSB was observed for calculations that use the crystallography-based structure. CONCLUSIONS: For specific DNA-protein complexes, our calculations can predict the protein radiolytic footprints on DNA. They show the significant contribution of the protein-induced DNA conformational change to DNA protection.

Bacterial Proteins↗
PubMed1978

How lac repressor recognizes lac operator.

Nucleotide analogs were substituted for unmodified nucleotides at specific sites in the lac operator sequence by a combination of chemical and enzymatic procedures. The nitrocellulose filter assay was used to study the interactions of these modified operators with wild-type (SQ) and tight-binding (QX86) lac repressors. These studies implicate directly the 5 methyl of thymine and the 2 amino of guanine as important operator-repressor contact sites. Furthermore, when these findings are combined with published results from other laboratories, a model for the lac operator-lac repressor interaction can be derived. Two important postulates follow from this model. (i) The repressor interacts at specific and defined sites with the N7 of guanine, the 5 methyl of thymine, the 2 amino of guanine, and the central major groove of the operator. (ii) The repressor binds to one side of the operator.

Base Sequence↗
PubMed1997

Escherichia coli lac repressor-lac operator interaction and the influence of allosteric effectors.

The wild type E. coli lac operator is embedded in a 35 base-pair DNA sequence containing extensive 2-fold symmetry, suggesting a symmetric repressor operator complex. However, deviations from strict 2-fold symmetry occur at the central base-pair and at three additional base-pairs. Using an operator fragment binding analysis we have determined: (a) a relative contribution each pair provides to the lac repressor-lac operator DNA complex, (b) the operator DNA length necessary for maximum binding to lac repressor; and (c) the contribution of the several non-symmetric base in the wild-type operator to the binding affinity. Since lac repressor-lac operator DNA interaction is reduced upon binding of the gratuitous inducer, isopropyl-beta-D-galactoside (IPTG), the same DNA fragment binding analysis was performed with the low affinity form of lac repressor. In the presence of inducer, the affinity for the left half site of the wild-type lac operator is reduced without significant reduction on the right half of the operator. Conversely, the anti-inducer orthonitrophenylfucoside (ONPF) which stabilizes the lac repressor-lac operator complex increases the binding affinity, particularly to the right half of the operator.

Allosteric Regulation↗
PubMed1979

Studies on gene control regions X. The effect of specific adenine-thymine transversions on the lac repressor-lac operator interaction.

Chemical and enzymatic methods were used to synthesize a transition (AT to GC) and a transversion (AT to TA) at a lac operator site known to interact with lac repressor through the thymine 5 methyl group. These operators also contained a poly(dA) . poly(dT) tail 8 to 12 base pairs in length at one end. Results suggest that the steric constraints of lac repressor relative to the position of the 5 methyl group are quite critical. For example a seven fold reduction in stability was observed for the transversion. Results also suggest that the operator spans at least 21 base pairs.

Adenine↗
PubMed1991

The roles of residues 5 and 9 of the recognition helix of Lac repressor in lac operator binding.

We constructed expression libraries for Lac repressor mutants with amino acid exchanges in positions 1, 2, 5 and 9 of the recognition helix. We then analysed the interactions of residues 5 and 9 with operator variants bearing single or multiple symmetric base-pair exchanges in positions 3, 4 and 5 of the ideal fully symmetric lac operator. We isolated 37 independent Lac repressor mutants with five different amino acids in position 5 of the recognition helix that exhibit a strong preference for particular residues in position 2 and, to a lesser extent, in position 1 of the recognition helix. Our results suggest that residue 5 of the recognition helix (serine 21) contributes to the specific recognition of base-pair 4 of the lac operator. They further suggest that residue 9 of the recognition helix (asparagine 25) interacts non-specifically with a phosphate of the DNA backbone, possibly between base-pairs 2 and 3.

Amino Acid Sequence↗
PubMed1987

Lac repressor-Lac operator complexes. Solution X-ray scattering and electrophoretic studies.

Complexes between the Lac repressor and a small DNA operator fragment (29 base pairs) were investigated using polyacrylamide gel electrophoresis and solution X-ray scattering. Titration of the DNA fragment with the repressor, followed by gel electrophoresis showed that only two types of complexes are formed with repressor/operator ratios of 0.5 and 2. Radii of gyration and forward scattered intensities were obtained from Guinier plots for repressor/operator ratios ranging from 0.3 to 2. They demonstrated that the first complex contains one repressor and two operators, whereas the second one contains four repressors and two operators. Mixing operator and repressor in equimolar concentrations leads to a mixture of both complexes. A possible model for the four repressor/two operator complex is proposed.

Base Sequence↗
PubMed1987

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.

Amino Acid Sequence↗
PubMed1983

NMR study of the interaction between the lac repressor and the lac operator.

Binding of the lac repressor headpiece, the N-terminal region of the lac repressor, to the lac operator of Escherichia coli was studied by 1H-NMR spectroscopy. Two DNA fragments, of 51 base pairs and 62 base pairs, containing the lac operator region, were investigated. The signals of their hydrogen-bonded imino protons were well resolved in the 500-MHz NMR spectra. The spectra of the free lac operator DNA are similar to those obtained from ring-current-shift calculations for a B-DNA structure. Complex formation with the headpiece led to small but nevertheless characteristic changes in the spectra. The fact that very few imino resonances shifted upon addition of headpiece, as well as the variety in direction and size of these chemical shifts, indicate the formation of a specific complex between the lac repressor and the lac operator. The observed changes in the resonance positions exclude the intercalation of tyrosine residues of the headpiece between adjacent base pairs of the lac operator as well as the formation of a cruciform structure. They rather reflect a small conformational transition in the DNA itself, caused for example by an alteration in the tilt of a few base pairs or a shift of the keto-enol tautomeric equilibrium of the bases towards the enolic form.

Bacterial Proteins↗
PubMed1988

Photo-CIDNP study of the interaction between lac repressor headpiece and lac operator DNA.

Lac repressor headpiece (HP) and intact lac repressor have been studied using the photo-CIDNP method. At neutral pH histidine 29, tyrosines 7, 12 and 17 and methionine 1 are polarised. His-29 polarizations are weaker and broader in HP59 than in HP51 indicating that the C-terminal octapeptide in HP59 adopts a conformation that allows an interaction with His-29. The photo-CIDNP spectra of intact lac repressor and HP51 are very similar, showing that the same residues are accessible to the photo-excited flavin. An equimolar mixture of HP51 and a 14 base pair lac operator fragment strongly suppresses the photo-CIDNP effect of tyrosines 7 and 17 and abolishes the His-29 polarizations. The results are compared with earlier photo-CIDNP measurements on a complex of headpiece with poly[d(AT)] and with a model derived from a 2D NMR study on a lac headpiece-operator complex.

DNA, Bacterial↗
PubMed1981

High-resolution nuclear magnetic resonance studies of the Lac repressor. 3. Unfolding of the Lac repressor headpiece.

At temperatures below 20 degrees C, the lac repressor headpiece (N-terminal amino acids 1--51) has a well-defined structure which is independent of ionic strength. Its unfolding with increasing temperature proceeds gradually with a characteristic transition temperature which depends on ionic strength. Unfolding has been studied by using NMR and CD. Shifts of several methyl and all of the tyrosyl resonances can be followed, allowing a detailed analysis of the temperature denaturation. At high ionic strength (1 M), the unfolding is complete at 85 degrees C, while at low ionic strength (0.01 M), it is complete by 65 degrees C. Native and partially unfolded structures are in rapid exchange during the unfolding, and the process appears completely reversible at all ionic strengths.

Circular Dichroism↗