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.     

Macromolecular binding equilibria in the lac repressor system: studies using high-pressure fluorescence spectroscopy

High hydrostatic pressure coupled with fluorescence polarization has been used to investigate protein subunit interactions and protein-operator association in lac repressor labeled with a long-lived fluorescent probe. On the basis of observation of a concentration-dependent sigmoidal decrease in the dansyl fluorescence polarization, we conclude that application of high hydrostatic pressure results in dissociation of the lac repressor tetramer. The 2-fold decrease in the rotational relaxation time and the high-pressure plateau are consistent with a tetramer to dimer transition. The volume change for tetramer dissociation to dimer is -82 +/- 5 mL/mol. The dissociation constant calculated from the data taken at 4.5 degrees C is 4.3 +/- 1.3 nM. The tetramer dissociation constant increases by a factor of 3 when the temperature is raised from 4.5 to 21 degrees C. A very small effect of inducer binding on the subunit dissociation is observed at 4.5 degrees C; the Kd increases from 4.5 to 7.1 nM. At 21 degrees C, however, inducer binding stabilizes the tetramer by approximately 0.8 kcal/mol. Pressure-induced monomer formation is indicated by the curves obtained upon raising the pH to 9.2. The addition of IPTG shifts the pressure transition to only slightly higher pressures at this pH, indicating that the stabilization of the tetramer by inducer is not as marked as that observed at pH 7.1. From the decrease in the polarization of the dansyl repressor-operator complexes, we also conclude that the application of pressure results their dissociation and that the volume change is large in absolute value (approximately 200 mL/mol). The lac repressor-operator complex is more readily dissociated upon the application of pressure than the tetramer alone, indicating that operator binding destabilizes the lac repressor tetramer.     

Heterogeneity of the two tryptophanyl residues in the lac repressor of Escherichia coli.

The wild-type lac repressor of Escherichia coli is a tetrameric protein which contains two tryptophanyl residues per subunit at positions 190 and 209. Solute perturbation studies of the tryptophan fluorescence of the repressor were performed using a polar but uncharged quencher, acrylamide, to prevent possible bias caused by ionic quenchers. The results indicate that the two tryptophan residues have different accessibilities to the quencher. In addition, contrary to a previous report, the accessibility of these tryptophan residues is not altered by isopropyl-β-d-thiogalactoside (IPTG) binding to the repressor. Similar studies with mutant lac repressor containing only a single tryptophan either at positions 190 or 209 suggest that tryptophan 209 is located in a region which is perturbed by inducer binding. That the two tryptophanyl residues have heterogeneous environments was further confirmed by nanosecond fluorescence spectroscopy which showed the wild-type lac repressor exhibiting two excited-state lifetimes, τ₁ = 5.3 ns and τ₂ = 10 ns. In the presence of 10^?3m IPTG, only a single lifetime of 6 ns was observed for the wild-type repressor suggesting that the inducer perturbs the tryptophan residue with the longer lifetime but not the one with the shorter lifetime. This is in accord with the observation that the mutant repressor containing only tryptophan 190 (the Tyr-209 repressor) has a single lifetime of 4.5 ns which is not altered by IPTG binding. The surprising finding that the mutant repressor which contains only tryptophan 209 (the Tyr-190 repressor) shows two excited-state lifetimes has been interpreted to indicate that the repressor either does not exhibit fourfold symmetry in its subunit arrangement or is present in two different conformational states.     

Engineered Tet repressor mutants with single tryptophan residues as fluorescent probes. Solvent accessibilities of DNA and inducer binding sites and interaction with tetracycline

Mutants of the Tn10-encoded Tet repressor containing single or no tryptophan residues were constructed by oligonucleotide-directed mutagenesis. The Trp-75 to Phe exchange reduces the dissociation rate of the complex with the inducer tetracycline by a factor of 2. The Trp-43 to Phe exchange has no effect on inducer binding. The fluorescence emission spectra of both tryptophan residues are quenched to a different extent by binding of tetracycline: Trp-75 is quenched to zero and Trp-43 to only 50%. It is concluded that Trp-75 is in the vicinity of the inducer binding site. The different fluorescence emission spectra of both tryptophan residues depend on the native structure of Tet repressor. Quenching studies with iodide indicate that the DNA binding motif is solvent exposed in free repressor and moves towards the interior of the protein upon inducer binding. The inducer binding site is in the interior of the protein. The fluorescence of tetracycline is enhanced upon binding to Tet repressor. The excitation at 280 nm results mainly from the change in environment and in part from energy transfer from tryptophan to the drug.     

Analysis of trp repressor-operator interaction by filter binding.

A filter binding assay was developed that allows measurement of specific binding of trp repressor to operator DNA. The most important feature of this procedure is the concentration and type of salt present in the binding buffer. Using this assay the dissociation constant of the repressor-operator complex was determined to be 2.6 X 10(-9) M, and 1.34 repressor dimers were found to be bound to each operator-containing DNA molecule. These values agree with those obtained by more complex methods. The dissociation constant of the repressor for the corepressor L-tryptophan in the presence of operator DNA was shown to be 2.5 X 10(-5) M. A synthetic 48 bp operator fragment was used to determine the repressor-operator dissociation constant in the presence of tryptophan or tryptophan analogs which have higher or lower affinities for aporepressor. The rate of dissociation of repressor from operator DNA also was determined. Our findings indicate that dissociation is influenced by the concentration of tryptophan or tryptophan analogs and suggest that release of the corepressor may be the first step in dissociation of the repressor-operator complex.     

A mutant lactose repressor with altered inducer and operator binding parameters

The lactose repressor protein from the mutant Escherichia coli BG185 contains valine at position 81 instead of alanine. Spectroscopic, chemical and direct binding measurements demonstrate that the BG185 protein exhibits properties similar to the wild-type repressor-inducer complex. Kinetic measurements of inducer binding to BG185 repressor yielded rate constants that were more than two orders of magnitude smaller than those observed for wild-type repressor; these results suggest that the structural transitions required for inducer binding are markedly impaired by the mutation. The fluorescence spectral shift in response to inducer binding was identical for mutant and wild-type proteins. This identity indicates direct effects of inducer binding on the tryptophan(s) near the sugar binding site rather than environmental changes consequent to conformational shifts. Analogy to the bacterial sugar binding proteins suggest that the Ala to Val change at position 81 in BG185 repressor yields a molecule that is fixed in a closed, sugar-binding conformation.     

Local mobility of the lac repressor molecule

The rotational mobility of lac repressor from Escherichia coli was investigated by nanosecond fluorescence depolarization spectroscopy. A single rotational correlation time (φ) of the repressor was observed by monitoring the emission anisotropic decay of the intrinsic tryptophan fluorescence. The small value of φ (9·5 ns) suggests that one or both of the two tryptophan residues in the repressor are located in a flexible segment of the protein molecule. This segmental flexibility is enhanced by binding of inducer (isopropyl-β-d-thiogalactoside) to the repressor while it is restrained by binding of anti-inducer (glucose) or small DNA fragments, as indicated by the changes in φ. Further time-dependent emission anisotropy studies with an extrinsic fluorescent probe, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate, covalently attached to the repressor yielded two rotational correlation times. The shorter φ_S (6·7 ns) also corresponds to a segmental flexibility whereas the longer φ_L (118 ns) represents the rotational motion of the entire repressor molecule. Both the values of φ_S and φ_L vary by addition of inducer or anti-inducer in a manner similar to that observed for the intrinsic tryptophan fluorescence but they are insensitive to addition of DNA fragments. The changes in local mobility of the lac repressor molecule observed in these studies may provide some insight into how inducer (or anti-inducer) destabilizes (or stabilizes) the repressor-operator complex.     

Allosteric regulation of inducer and operator binding to the lactose repressor

The effects of cysteine modification and variations in pH on the equilibrium parameters for inducer and operator binding to the lactose repressor protein were examined. Operator binding affinity was minimally affected by increasing the pH from 7.5 to 9.2, whereas inducer binding was decreased for both the unliganded protein and the repressor-operator complex over the same range. Inducer binding to the repressor became more cooperative at high pH. The midpoint for the change in inducer affinity and cooperativity was pH 8.3; this value correlates well with cysteine ionization. The differential between repressor-operator affinity in the presence and absence of inducer was significantly decreased by modification of the protein with methyl methanethiosulfonate (MMTS). In contrast to unreacted protein, the inducer binding parameters for MMTS-modified repressor were largely unaffected by pH variation. The free energy for formation of the completely liganded protein was calculated for two pathways; the delta G values for these two independent routes were equivalent only for stoichiometries of four inducers and two operators per repressor molecule. All of the binding data were analyzed quantitatively by using a Monod-Wyman-Changeux two-state model for allosteric regulation. The observed dependences of the isopropyl beta-D-thiogalactoside binding curves on pH, DNA concentration, and MMTS modification were fitted by varying only the equilibrium constant between the two conformational states of the protein. With this analysis, high pH favors the T (high operator/low inducer affinity) state, while modification of cysteine-281 with MMTS elicits a shift into the R (high inducer/low operator affinity) state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Repressor--operator interaction in the lac operon. II. Observations at the tyrosines and tryptophans

This paper shows that ¹⁹F-nuelear magnetic resonance spectroscopy on 3-fluoro-tyrosine and 5-fluorotryptophan-substituted wild-type lactose operon repressors from Escherichia coli can be used to examine the interactions with lac operator DNA.A survey of inducer and salt concentration effects on the repressor-operator complex is presented. The data lead us to a scheme for the interactions between the repressor, operator and inducer, in both binary and ternary complexes, that accommodate the results published by others.The complex between the tetrameric repressor and one 36 base-pair operator DNA fragment results in the simultaneous broadening of the resonances from all four N-terminal DNA binding domains. The actual contacts made by these binding domains are similar but probably not identical.The binding of the inducer molecule to the tetrameric repressor results in an allosteric change that can be monitored by the increased intensity of the resonances from individual tyrosine residues in the N-terminal binding domain. This increased N-terminal tyrosine resonance intensity in the complex is transmitted to repressor subunits that have not yet bound an inducer molecule.     

Dissociation of the lactose repressor protein tetramer using high hydrostatic pressure

Dissociation of lac repressor tetramer by high hydrostatic pressures was monitored with intrinsic tryptophan fluorescence. With the assumption of complete dissociation to monomer, tryptophan polarization data gave delta V a approximately 170 mL/mol and the concentration for 50% tetramer dissociation, C1/2, was 3.8 X 10(-8) M. Upon addition of inducer, the calculated delta V a increased to approximately 220 mL/mol and the C1/2 decreased to approximately 1 X 10(-8) M, a free energy difference of approximately 0.7 kcal. These results indicate a modest stabilization of the tetramer by the presence of inducer. Monitoring the average energy of tryptophan emission demonstrated that tetramer dissociation takes place over the same range of pressures as evidenced by the polarization data and IPTG dissociation can be more or less superimposed upon tetramer dissociation depending upon the ligand concentration used. Although the two transitions cannot be separated entirely, the delta V a for the region of the pressure dependence dominated by ligand dissociation was 69 mL/mol, an unexpectedly large value. For tetramer modified with methyl methanethiosulfonate, subunit dissociation was shifted to much higher pressures and IPTG dissociation did not occur. The delta V a for subunit association was calculated as approximately 160 mL/mol, and the C1/2 was 3.5 X 10(-9) M. Interactions at the subunit interface of the modified protein are apparently stronger than in the unmodified protein. The absence of inducer dissociation from the MMTS-modified tetramer by the application of high hydrostatic pressure suggests that the volume change for inducer binding to the modified protein is much smaller than that observed for the unmodified repressor.     

Sequence-specific interactions of the tight-binding I12-X86 lac repressor with non-operator DNA.

The tight-binding I12-X86 lac repressor binds to non-operator DNA in a sequence-specific fashion. Using the DNA of the E. coli I gene we have investigated these sequence-specific interactions and compared them to the operator binding of wild-type repressor. The specific, non-operator DNA interactions are sensitive to the inducer IPTG. One strong binding site in the I gene DNA was found to be one of two expected on the basis of their homology with the lac operator. The binding of I12-X86 repressor to this site was visualized using the footprinting technique, and found to be consistent with an operator-like binding configuration. The protection pattern extends into an adjacent sequence suggesting that two repressor tetramers are bound in tandem.     

Ultraviolet radiation-induced derepression of the lactose operon of E. coli

The effect of ultraviolet irradiation of a regulatory protein, the lac repressor, on its interactions with operator DNA is investigated by spectroscopic and electrophoresis methods. A second set of experiments is performed to assay the capacity of the system containing the irradiated repressor to be induced by IPTG. The protein-nucleic acid interactions are modified upon ultraviolet irradiation of the repressor. The inducer becomes ineffective and repressor stays "locked" to DNA in conditions in which the native repressor is released from the system. These facts are discussed in terms of genes repression and of promotion step in ultraviolet induced carcinogenesis.     

Radiation disrupts protein-DNA complexes through damage to the protein. The lac repressor-operator system.

Eon, S., Culard, F., Sy, D., Charlier, M. and Spotheim-Maurizot, M. Radiation Disrupts Protein-DNA Complexes through Damage to the Protein. The lac Repressor-Operator System. Radiat. Res. 156, 110-117 (2001).Binding of a protein to its cognate DNA sequence is a key step in the regulation of gene expression. If radiation damage interferes with protein-DNA recognition, the entire regulation process may be perturbed. We have studied the effect of gamma rays on a model regulatory system, the E. coli lactose repressor-operator complex. We have observed the disruption of the complex upon irradiation in aerated solution. The complex is completely restored by the addition of nonirradiated repressor, but not by the addition of nonirradiated DNA. Thus radiation disrupts the DNA-protein complex by affecting the binding ability of the protein. This interpretation is supported by the dramatic loss of binding ability of a free irradiated repressor toward nonirradiated DNA. Interestingly, the dose necessary for the disruption of the irradiated complex is higher than that for inducing the complete loss of the binding ability of the free irradiated repressor. This may be due to the protection of key amino acids by the bound DNA. As seen from calculations of the accessibility of amino acids to radiolytic OH(.), the protection is due to both masking and conformational effects.     

Comparing native and irradiated E. coli lactose repressor-operator complex by molecular dynamics simulation

The function of the E. coli lactose operon requires the binding of the tetrameric repressor protein to the operator DNA. We have previously shown that γ-irradiation destabilises the repressor-operator complex because the repressor gradually loses its DNA-binding ability (Radiat Res 170:604–612, 2008). It was suggested that the observed oxidation of tyrosine residues and the concomitant structural changes of irradiated headpieces (DNA-binding domains of repressor monomers) could be responsible for the inactivation. To unravel the mechanisms that lead to repressor-operator complex destabilisation when tyrosine oxidation occurs, we have compared by molecular dynamic simulations two complexes: (1) the native complex formed by two headpieces and the operator DNA, and (2) the damaged complex, in which all tyrosines are replaced by their oxidation product 3,4-dihydroxyphenylalanine (DOPA). On a 20 ns time scale, MD results show effects consistent with complex destabilisation: increased flexibility, increased DNA bending, modification of the hydrogen bond network, and decrease of the positive electrostatic potential at the protein surface and of the global energy of DNA-protein interactions.