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.     

Radiation abolishes inducer binding to lactose repressor

The lactose operon functions under the control of the repressor-operator system. Binding of the repressor to the operator prevents the expression of the structural genes. This interaction can be destroyed by the binding of an inducer to the repressor. If ionizing radiations damage the partners, a dramatic dysfunction of the regulation system may be expected. We showed previously that gamma irradiation hinders repressor-operator binding through protein damage. Here we show that irradiation of the repressor abolishes the binding of the gratuitous inducer isopropyl-1-beta-D-thiogalactoside (IPTG) to the repressor. The observed lack of release of the repressor from the complex results from the loss of the ability of the inducer to bind to the repressor due to the destruction of the IPTG binding site. Fluorescence measurements show that both tryptophan residues located in or near the IPTG binding site are damaged. Since tryptophan damage is strongly correlated with the loss of IPTG binding ability, we conclude that it plays a critical role in the effect. A model was built that takes into account the kinetic analysis of damage production and the observed protection of its binding site by IPTG. This model satisfactorily accounts for the experimental results and allows us to understand the radiation-induced effects.     

Uracil-DNA glycosylase as a probe for protein--DNA interactions.

The DNA repair enzyme Uracil-DNA Glycosylase (UDG) can be used to investigate three different features of protein-DNA interactions. Complexes can be probed by simple protection experiments ('footprinting') or by two kinds of interference assays: a missing thymine site (MT-site) experiment and a missing thymine methyl site (MTM-site) experiment. The three probing methods are assessed using the well-characterized in vitro systems of lambda repressor and lac repressor binding to their respective operator sites. The results obtained with UDG probing agree well with previous probing experiments on the same systems and, in certain cases, extend previous interpretations: for example, comparison of the results obtained with the two interference assays shows that formation of the lac repressor-operator complex requires interactions with the methyl group of one particular thymine residue (T-13) in the operator but also requires interactions with other parts of the thymine base at operator positions 7, 8, 9, 21, 23 and 24. Overall, the properties of UDG recommend it as a versatile and convenient method to investigate DNA-protein interactions both in vitro and possibly in vivo.     

Coupling of protein assembly and DNA binding: biotin repressor dimerization precedes biotin operator binding

The kinetics of coupling of protein dimerization and DNA binding have been investigated in the biotin repressor system. Two repressor monomers bind to the 40 base-pair biotin operator sequence. In previous analyses of equilibrium-binding data the weak dimerization of the repressor has justified using a model in which two protein monomers bind cooperatively to the operator site. Here, rapid kinetic methods have been used to directly determine the binding mechanism. Results of rapid-mixing DNaseI footprinting measurements of association of the repressor with operator indicate that the binding process involves at least two steps. Results of measurements of the unimolecular dissociation of the complex reveal a half-life of approximately 400 seconds. Analysis of the data using a combination of simulation and global non-linear least-squares analysis provides support for a binding model in which a preformed repressor dimer associates with the biotin operator. This kinetic model is consistent with the previously proposed model for regulation of the functional switch in the repressor from enzyme to site-specific DNA-binding protein.     

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.     

The CTXphi repressor RstR binds DNA cooperatively to form tetrameric repressor-operator complexes

CTX is a filamentous bacteriophage that encodes cholera toxin and integrates into the Vibrio cholerae genome to form stable lysogens. In CTX lysogens, gene expression originating from the rstA phage promoter is repressed by the phage-encoded repressor RstR. The N-terminal region of RstR contains a helix-turn-helix DNA-binding element similar to the helix-turn-helix of the cI/Cro family of phage repressors, whereas the short C-terminal region is unrelated to the oligomerization domain of cI repressor. Purified His-tagged RstR bound to three extended 50-bp operator sites in the rstA promoter region. Each of the RstR footprints exhibited a characteristic staggered pattern of DNase I-accessible regions that suggested RstR binds DNA as a dimer-of-dimers. In gel permeation chromatography and cross-linking experiments, RstR oligomerized to form dimers and tetramers. RstR was shown to be tetrameric when bound to operator DNA by performing mobility shift experiments with mixtures of RstR and a lengthened active variant of RstR. Binding of RstR to the high affinity O1 site could be fit to a cooperative model of operator binding in which two RstR dimers associate to form tetrameric RstR-operator complexes. The binding of RstR dimers to the left or right halves of O1 operator DNA was not observed in mobility shift assays. These observations support a model in which protein-protein contacts between neighboring RstR dimers contribute to strong operator binding.     

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.     

Ligand-linked structural changes in the Escherichia coli biotin repressor: the significance of surface loops for binding and allostery.

The Escherichia coli repressor of biotin biosynthesis (BirA) is an allosteric site-specific DNA-binding protein. BirA catalyzes synthesis of biotinyl-5'-AMP from substrates biotin and ATP and the adenylate serves as the positive allosteric effector in binding of the repressor to the biotin operator sequence. Although a three-dimensional structure of the apo-repressor has been determined by X-ray crystallographic techniques, no structures of any ligand-bound forms of the repressor are yet available. Results of previously published solution studies are consistent with the occurrence of conformational changes in the protein concomitant with ligand binding. In this work the hydroxyl radical footprinting technique has been used to probe changes in reactivity of the peptide backbone of BirA that accompany ligand binding. Results of these studies indicate that binding of biotin to the protein results in protection of regions of the central domain in the vicinity of the active site and the C-terminal domain from chemical cleavage. Biotin-linked changes in reactivity constitute a subset of those linked to adenylate binding. Binding of both bio-5'-AMP and biotin operator DNA suppresses cleavage at additional sites in the amino and carboxy-terminal domains of the protein. Varying degrees of protection of the five surface loops on BirA from hydroxyl radical-mediated cleavage are observed in all complexes. These results implicate the C-terminal domain of BirA, for which no function has previously been known, in small ligand and site-specific DNA binding and highlight the significance of surface loops, some of which are disordered in the apoBirA structure, for ligand binding and transmission of allosteric information in the protein.     

Computer simulations and experimental studies of gel mobility patterns for weak and strong non-cooperative protein binding to two targets on the same DNA: application to binding of tet repressor variants to multiple and single tet operator sites

A series of computer simulations of gel patterns assuming non-cooperative binding of a protein to two targets on the same DNA fragment was performed and applied to interprete gel mobility shift experiments of Tet repressor-tet operator binding. While a high binding affinity leads to the expected distribution of free DNA, DNA bound by one repressor dimer and DNA bound by two repressor dimers, a lower affinity or an increased electrophoresis time results in the loss of the band corresponding to the singly occupied complex. The doubly occupied complex remains stable under these conditions. This phenomenon is typical for protein binding to DNA fragments with two identical sites. It results from statistical disproportionation of the singly occupied complex in the gel. The lack of the singly occupied complex is commonly taken to indicate cooperative binding, however, our analysis shows clearly, that cooperativity is not needed to interprete these results. Tet repressor proteins and small DNA fragments with two tet operator sites have been prepared from four classes of tetracycline resistance determinants. The results of gel mobility shift analyses of various complexes of these compounds confirm the predictions. Furthermore, calculated gel patterns assuming different gel mobilities of the two singly occupied complexes show discrete bands only if the electrophoresis time is shorter than the inverse of the microscopic dissociation rate constant. Simulations assuming increasing dissociation rates predict that the two bands first merge into one, which then disappears. This behavior was verified by gel mobility analyses of Tet repressor-tet operator titrations at increased salt concentrations as well as by direct footprinting of the complexes in the gel. It is concluded that comparison of the intensities of the single and the double occupation bands allow a rough estimation of the dissociation rate constant. On this basis the sixteen possible Tet repressor-tet operator combinations can be ordered with decreasing binding affinities by a simple gel shift experiment. The implications of these results for gel mobility analyses of other protein-DNA complexes are discussed.     

Characterization of the arginine repressor from Salmonella typhimurium and its interactions with the carAB operator.

Primer extension experiments showed that the argR gene, encoding the arginine repressor in Salmonella typhimurium, is transcribed from a single promoter that is negatively regulated by arginine. A repressor overproducing strain was constructed and the repressor was purified to homogeneity. Gel filtration, sedimentation and cross-linking studies established that the native repressor is a hexamer of identical 17,000 Mr subunits. Gel retardation experiments indicate that the apparent dissociation constant for repressor/carAB operator is 6 x 10(-12) M. These experiments showed that arginine is essential for binding of the repressor to the DNA and that pyrimidine nucleotides have no significant effect on this binding. These results indicate that the effect of pyrimidines on expression of the arginine sensitive "downstream" carAB promoter is not directly mediated by the arginine repressor. These experiments also suggest that a single hexamer binds to the carAB operator, which carries two previously defined "ARG box" sequences that characterize operators for arg genes. Gel retardation experiments with DNA fragments carrying the individual ARG boxes showed that both boxes are required for effective binding of the hexameric repressor to the operator, indicating that the ARG boxes comprise a single binding site for the repressor. Analysis of the potential secondary structure of the arginine repressor does not reveal any of the recognizable structural motifs common to a number of DNA-binding proteins. A combination of DNase I, premethylation interference, depurination and hydroxyl radical footprinting techniques were employed to characterize the interactions of the repressor with the carAB operator, with the results suggesting that the repressor predominantly interacts with A.T residues in this region. Comparative DNA sequence analysis of the known arginine operators of enteric bacteria further indicates that the specificity of interaction may be based more on the precise distance between two defined A.T-rich regions rather than on the specific nucleotide sequence.     

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.     

How Trp repressor binds to its operator.

We propose that the generally accepted model of a single Trp repressor dimer binding to a center of symmetry in the natural trp operator (Otwinowski et al., 1988) is wrong. We show here that the Trp repressor binds to a sequence whose center is located four base pairs either to the right or to the left of the central axis of symmetry that was previously identified. We show that: (i) the oligonucleotide used by Otwinowski et al. is not retarded by the Trp repressor in a mobility shift assay under conditions wherein a shorter oligonucleotide carrying our consensus sequence is retarded, (ii) that methylation protection experiments on the full natural operator sequence and the short oligonucleotide protect similar patterns and (iii) that by varying every base in the shorter oligonucleotide, we can demonstrate an optimal sequence for Trp repressor binding.     

Binding of the arginine repressor of Escherichia coli K12 to its operator sites.

In the arginine regulon of Escherichia coli K12 each of the eight operator sites consists of two 18-base-pair-long palindromic sequences called ARG boxes. In the operator sites for the structural genes of the regulon the two ARG boxes are separated by three base-pairs, in the regulatory gene argR they are separated by two base-pairs. The hexameric arginine repressor, the product of argR, binds to the two ARG boxes in an operator in the presence of L-arginine. From the results of various kinds of in vitro footprinting experiments with the ARG boxes of argF and argR (DNase I protection, hydroxyl radical, ethylation and methylation interference, methylation protection) it can be concluded that: (1) the repressor binds simultaneously to two adjacent ARG boxes; (2) that it binds on one face of the double helix; and (3) that it forms contacts with the major and minor grooves of each ARG box, but not with the central three base-pairs. The repressor can bind also to a single ARG box, but its affinity is about 100-fold lower than for two ARG boxes. From gel retardation experiments with 3H-labeled repressor and 32P-labeled argF operator DNA, it is concluded that the retarded DNA-protein complex contains no more than one repressor molecule per operator site and that most likely one hexamer binds to two ARG boxes. The bound repressor was shown to induce bending of argF operator DNA. The bending angle calculated from the results of gel retardation experiments is about 70 degrees and the bending center was located within the region encompassing the ARG boxes. The main features that distinguish the arginine repressor from other repressors studied in E. coli are its hexameric nature and the simultaneous binding of one hexameric molecule to two palindromic ARG boxes that are close to each other.     

Proton-linked contributions to site-specific interactions of lambda cI repressor and OR

The effects of proton activity on the site-specific interactions of cI repressors with operator sites OR were studied by using DNase I footprint titration. Individual-site binding isotherms were obtained for the binding of repressor to each site of wild-type OR and of mutant operators in which binding to some sites is eliminated. The Gibbs energies for binding and for cooperativity (in every operator configuration) were determined at each pH (range 5-8). The proton-linked effects clearly account for a significant fraction of the difference in affinities for the three operator sites. The most dramatic effects on the repressor-operator binding interactions are at acid pH, and therefore do not involve the basic groups in the repressor N-terminal arm known to contact the DNA. Also, the proton-linked effects are different at the three operator sites as indicated by significantly different derivative relationships, partial derivative of ln k versus partial derivative of ln aH = net proton absorption (delta nu bar(H)). These results implicate ionizable repressor groups which may not contact the DNA and conformational differences between the three repressor-operator site complexes as being important components to the mechanism of site specificity. The extensive data base generated by these studies was also used to reevaluate the traditional models used to describe cooperativity in this system. The results confirm the lack of significant cooperative interaction between OR1 and OR3 at all conditions. However, the data for some experimental conditions are clearly inconsistent with the (selection) rule, that cooperative interaction between OR2 and OR3 is eliminated by ligation at OR1.