Lac Repressor-operator System Research Articles

High-resolution specificity from DNA sequencing highlights alternative modes of Lac repressor binding.

Knowing the specificity of transcription factors is critical to understanding regulatory networks in cells. The lac repressor-operator system has been studied for many years, but not with high-throughput methods capable of determining specificity comprehensively. Details of its binding interaction and its selection of an asymmetric binding site have been controversial. We employed a new method to accurately determine relative binding affinities to thousands of sequences simultaneously, requiring only sequencing of bound and unbound fractions. An analysis of 2560 different DNA sequence variants, including both base changes and variations in operator length, provides a detailed view of lac repressor sequence specificity. We find that the protein can bind with nearly equal affinities to operators of three different lengths, but the sequence preference changes depending on the length, demonstrating alternative modes of interaction between the protein and DNA. The wild-type operator has an odd length, causing the two monomers to bind in alternative modes, making the asymmetric operator the preferred binding site. We tested two other members of the LacI/GalR protein family and find that neither can bind with high affinity to sites with alternative lengths or shows evidence of alternative binding modes. A further comparison with known and predicted motifs suggests that the lac repressor may be unique in this ability and that this may contribute to its selection.

Lac-regulated system for generating adenovirus 5 vaccine vectors expressing cytolytic human immunodeficiency virus 1 genes

Adenovirus (Ad) vectors have been developed as human immunodeficiency-1 (HIV-1) vaccine vectors because they consistently induce immune responses in preclinical animal models and human trials. Strong promoters and codon-optimization are often used to enhance vaccine-induced HIV-1 gene expression and immunogenicity. However, if the transgene is inherently cytotoxic in the cell line used to produce the vector, and is expressed at high levels, it is difficult to rescue a stable Ad HIV-1 vaccine vector. Therefore we hypothesized that generation of Ad vaccine vectors expressing cytotoxic genes, such as HIV-1 env, would be more efficient if expression of the transgene was down-regulated during Ad rescue. To test this hypothesis, a Lac repressor–operator system was applied to regulate expression of reporter luciferase and HIV-1 env transgenes during Ad rescue. The results demonstrate that during Ad rescue, constitutive expression of the Lac repressor in 293 cells reduced transgene expression levels to approximately 5% of that observed in the absence of regulation. Furthermore, Lac-regulation translated into more efficient Ad rescue compared to traditional 293 cells. Importantly, Ad vectors rescued with this system showed high levels of transgene expression when transduced into cells that lack the Lac repressor protein. The Lac-regulated system also facilitated the rescue of modified Ad vectors that have non-native receptor tropism. These tropism-modified Ad vectors infect a broader range of cell types than the unmodified Ad, which could increase their effectiveness as a vaccine vector. Overall, the Lac-regulated system described here (i) is backwards compatible with Ad vector methods that employ bacterial-mediated homologous recombination, (ii) is adaptable for the engineering of tropism-modified Ad vectors, and (iii) does not require co-expression of regulatory genes from the vector or the addition of exogenous chemicals to induce or repress transgene expression. This system therefore could facilitate the development of Ad-based vaccine candidates that otherwise would not be feasible to generate.

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.

African swine fever virus structural protein pE120R is essential for virus transport from assembly sites to plasma membrane but not for infectivity.

This report examines the role of African swine fever virus (ASFV) structural protein pE120R in virus replication. Immunoelectron microscopy revealed that protein pE120R localizes at the surface of the intracellular virions. Consistent with this, coimmunoprecipitation assays showed that protein pE120R binds to the major capsid protein p72. Moreover, it was found that, in cells infected with an ASFV recombinant that inducibly expresses protein p72, the incorporation of pE120R into the virus particle is dependent on p72 expression. Protein pE120R was also studied using an ASFV recombinant in which E120R gene expression is regulated by the Escherichia coli lac repressor-operator system. In the absence of inducer, pE120R expression was reduced about 100-fold compared to that obtained with the parental virus or the recombinant virus grown under permissive conditions. One-step virus growth curves showed that, under conditions that repress pE120R expression, the titer of intracellular progeny was similar to the total virus yield obtained under permissive conditions, whereas the extracellular virus yield was about 100-fold lower than in control infections. Immunofluorescence and electron microscopy demonstrated that, under restrictive conditions, intracellular mature virions are properly assembled but remain confined to the replication areas. Altogether, these results indicate that pE120R is necessary for virus dissemination but not for virus infectivity. The data also suggest that protein pE120R might be involved in the microtubule-mediated transport of ASFV particles from the viral factories to the plasma membrane.

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.

Protein binding protects sites on stable episomes and in the chromosome from de novo methylation.

We have utilized the Escherichia coli lac repressor-operator system to test whether protein binding can interfere with de novo DNA methylation in mammalian cells. We find that a DNA binding protein can protect sites on the episome as well as in the genome from the de novo methylation activity of Dnmt3a. Transcriptional machinery moving through the binding sites does not affect the de novo methylation of these sites, and it does not affect the binding protein protection of these sites from de novo methylation. This study and previous studies provide a possible mechanism for the observation that an Sp1 site can serve as a cis-acting signal for demethylation and for preventing de novo methylation of the CpG island upstream of the mouse adenine phosphoribosyltransferase (Aprt) gene. These findings also support the hypothesis that protein binding may play a crucial role in changes of CpG methylation pattern in mammalian cells.

Inducible gene expression from African swine fever virus recombinants: analysis of the major capsid protein p72.

A method to study the function of individual African swine fever virus (ASFV) gene products utilizing the Escherichia coli lac repressor-operator system has been developed. Recombinant viruses containing both the lacI gene encoding the lac repressor and a strong virus late promoter modified by the insertion of one or two copies of the lac operator sequence at various positions were constructed. The ability of each modified promoter to regulate expression of the firefly luciferase gene was assayed in the presence and in the absence of the inducer isopropyl beta-D-thiogalactoside (IPTG). Induction and repression of gene activity were dependent on the position(s) of the operator(s) with respect to the promoter and on the number of operators inserted. The ability of this system to regulate the expression of ASFV genes was analyzed by constructing a recombinant virus inducibly expressing the major capsid protein p72. Electron microscopy analysis revealed that under nonpermissive conditions, electron-dense membrane-like structures accumulated in the viral factories and capsid formation was inhibited. Induction of p72 expression allowed the progressive building of the capsid on these structures, leading to assembly of ASFV particles. The results of this report demonstrate that the transferred inducible expression system is a powerful tool for analyzing the function of ASFV genes.

High-level expression of human c-Src can cause a spherical morphology without loss of anchorage-dependent growth of NIH 3T3 cells

To investigate whether overexpression of human c-Src leads to cell rounding in anchorage-dependent NIH 3T3 fibroblasts, we have established c-Src-inducible cell lines using a lac repressor–operator system. RN1 cells, which expressed c-Src at a high level after induction, exhibited a spherical morphology and ceased to grow in monolayer culture. RN1 cells, however, exhibited neither focus-forming ability nor anchorage-independent growth potential with or without induction. Induced RN1 c-Src was phosphorylated at Ser 75, a previously reported spherical cell-associated site, and at Tyr 419. These data demonstrated for the first time that highly elevated human c-Src tyrosine kinase activity can cause NIH 3T3 cells to have a spherical morphology without loss of anchorage-dependent growth. The inducible cell line should be useful to study the mechanism for cell rounding by c-Src.

20] NMR and nucleic acid-protein interactions: The Lac repressor-operator system

This chapter discusses the lac repressor-operator system as an example to illustrate the nuclear magnetic resonance (NMR) approach to protein-DNA recognition. The recognition of DNA sequences, by proteins, lies at the heart of many cellular processes, such as gene regulation, replication and DNA repair. Therefore, the mechanism of protein-DNA interaction constitutes a major problem in molecular biology. Approaches to this problem range from biochemical and genetic studies (mutagenesis of DNA-binding proteins) to the application of the methods of structural biology (X-ray crystallography and NMR spectroscopy). NMR spectroscopy started to contribute around 1985, with the structure elucidation of the lac repressor headpiece and a low-resolution structure of the headpiece-operator complex in 1987, whereas in the first structures all contained a helix-turn-helix motif as the essential DNA-binding sub domain, later a plethora of DNA-binding motifs were characterized, including zinc-fingers, leucine zippers, helix-loop-helix proteins, and even B-sheet DNA-binding proteins. NMR has significantly contributed to this, as often DNA-binding domains of proteins are relatively small, independently folded domains that can be expressed and studied separately. For example, for the various subclasses of zinc-fingers, the first structural information came from NMR. The chapter concludes that the structure of the lac headpiece-operator complex agrees very well with the genetic data, and indeed provides a basis for a detailed interpretation of these data in structural terms.

Free DNA Concentration in E. coli Estimated by an Analysis of Competition for DNA Binding Proteins

Transcription in E. coli is often controlled by the binding of specific gene-regulatory proteins. Binding of these proteins to their specific DNA binding sites occurs in the presence of a large excess of "nonspecific" genomic DNA. Binding to a specific DNA site thus depends on the concentration of regulatory protein, on its affinities for specific and competing nonspecific binding sites, and on the free concentrations of those sites. Although it is probable that genomic DNA is largely occluded by protein binding or by condensation in vivo, the actual extent to which the DNA is available to act as a competitor for specific binding (i.e. the effective concentration of nonspecific DNA) is not known. Because many regulatory interactions occur simultaneously in a cell, it is reasonable to expect that they will have evolved to function at equilibrium with a shared concentration of competing nonspecific DNA. This premise was the basis for this study. In vitro binding data were compiled for six regulatory proteins that function in E. coli, and used to calculate theoretical equilibrium binding distributions. The calculated distributions were used to evaluate the regulatory states of promoters according to models based on the equilibrium occupancies of regulatory sites. For four proteins whose DNA-binding affinities are modulated by ligand binding (CAP, lac repressor, trp repressor and ara C), regulation was assessed as the extent to which the presence of the modulator could affect the occupancy by protein of the specific sites (e.g. the difference in equilibrium occupancy by CAP of CAP binding sites between conditions of high and low concentrations of CAP's affinity modulator, cAMP). For two proteins whose site affinities are not modulated by ligand binding (λ repressor and λ- cro), regulation was assessed by specific site occupancy at equilibrium. These regulation profiles were compared to determine whether a single concentration of nonspecific competing DNA is compatible with effective regulation as defined for all of the systems. For five of the six modeled systems (CAP, trp repressor, araC, λ repressor and λ- cro), a free nonspecific DNA concentration on the order of 10 -4 M base pairs is compatible with regulation based on equilibria of the protein-DNA interactions. The lac repressor-operator system is an exception to these results: as has been shown previously, the regulation of operator binding by low molecular weight inducers increases with increasing concentrations of nonspecific DNA (von Hippel et al., 1974 Proc. natn. Acad. Sci. U.S.A. 71, 4808-4812). This discrepancy may indicate that the regulatory state of the lac operator depends on the lifetime of the repressor-operator complex, not simply its equilibrium fractional occupancy. With this exception, the results based on equilibria for the other five systems are consistent with regulation of transcription by protein binding in the presence of an effective DNA concentration [∼ 10 -4 M base pairs (b.p.)] that is less than 1% of the absolute concentration of total genomic DNA.

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 19 F-NMR, three pairs of naturally occurring, symmetry related thymine residues (at +6/+16, +8/+14 and +1/+21) were substituted pair wise by 5-fluoro-deoxyuridines (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.

Gene expression in Bacillus brevis under control of the Lac repressor-operator system from Escherichia coli

Abstract We constructed two inducible plasmid vectors which were different at the replication origin to control the expression of heterologous genes in Bacillus brevis. The Escherichia coli lac repressor gene was expressed by using the promoter region of the cell wall protein (MWP) gene of B. brevis 47. The lac operator was placed downstream from the P2 promoter of the MWP gene. The gene of Bacillus licheniformis α-amylase (BLA) or for human salivary α-amylase (HAMY) was inserted immediately downstream of the lac operator. The production of BLA by B. brevis carrying the inducible plasmids pNU550BLA and pHT550BLA thus constructed was induced 8 and 100-fold, respectively, by the addition of isopropyl-β- d -thio-galactopyranoside (IPTG). The production of HAMY carrying the inducible plasmid pNU550HAMY was induced 6-fold by the addition of IPTG.

DNA looping alters local DNA conformation during transcription

The effect of protein-mediated DNA looping on local DNA conformation during active transcription was studied using the lac repressor-operator system. Our results suggest that lac repressor-mediated DNA looping within a plasmid DNA molecule containing two lac repressor binding sequences in vivo effectively separates plasmid DNA into two topological domains. Supercoils generated by transcription within each topological domain can be rapidly removed by DNA topoisomerase I.

Regulation of coliphage T3 and T7 RNA polymerases by the lac represser-operator system

The single-polypeptide RNA polymerases that are encoded by bacteriophage T7 and its relatives form the basis of highly specific and efficient transcription systems. Here, we describe the regulation of transcription from phage promoters by the lac repressor-operator system of Escherichia coli. A synthetic oligodeoxyribonucleotide that contains the core sequence of the lac operator ( lacO) was cloned at various distances downstream from the transcription start point ( tsp) of the T3 and T7 promoters. The ability of lac represser to prevent transcription from the phage promoters in vitro was dependent on the position of the operator. Efficient repression was observed when the center of the operator was placed between + 14 and + 27 (+ 1 being the tsp), whereas the repressor had little effect when bound to operators centered at + 64. For in vivo studies, the chloramphenicol acetyltransferase (CAT)-encoding reporter gene was placed under the control of various promoter-operator constructs, and introduced into bacterial cells containing the genes for the lac repressor and T3 or T7 RNA polymerase. As with in vitro studies, high levels of repression (> 4000-fold) of T3 and T7 RNA polymerase activity were achieved, and repression was reversed by the inducer isopropyl-β- d-thiogalactopyranoside. When the T3 promoter- lacO constructs are used to regulate the expression of a target gene in combination with an inducible RNA polymerase gene under control of the lacUV5 promoter, the doubly regulated system provides extremely tight levels of repression, yet allows high levels of expression after induction. In such a system, we observed a > 10 5-fold increase in CAT activity within 30 min after induction. This system should prove useful in cloning and expressing genes that are potentially toxic to the host cells.

Association kinetics with coupled diffusion. An extension to coiled-chain macromolecules applied to the LAC repressor-operator system

The association of a molecule onto a specified binding site on a large chain-like macromolecule is described in the “sliding” model, where the molecule is allowed to move along the chain in a one-dimensional diffusion which is coupled to the three-dimensional diffusion in solution. The present work extends a previous one by treating the chains more generally as coiled instead of straight. The model is applied to the lac repressor-operator association. A general expression for the rate of unspecific attachment to a chain-like macromolecule is also derived.