ABSTRACT
Natural prokaryotic gene repression systems often exploit DNA looping to increase the local concentration of gene repressor proteins at a regulated promoter via contributions from repressor proteins bound at distant sites. Using principles from the Escherichia coli lac operon we design analogous repression systems based on target sequence-programmable Transcription Activator-Like Effector dimer (TALED) proteins. Such engineered switches may be valuable for synthetic biology and therapeutic applications. Previous TALEDs with inducible non-covalent dimerization showed detectable, but limited, DNA loop-based repression due to the repressor protein dimerization equilibrium. Here, we show robust DNA loop-dependent bacterial promoter repression by covalent TALEDs and verify that DNA looping dramatically enhances promoter repression in E. coli. We characterize repression using a thermodynamic model that quantitates this favorable contribution of DNA looping. This analysis unequivocally and quantitatively demonstrates that optimized TALED proteins can drive loop-dependent promoter repression in E. coli comparable to the natural LacI repressor system. This work elucidates key design principles that set the stage for wide application of TALED-dependent DNA loop-based repression of target genes.
ABSTRACT
Architectural proteins alter the shape of DNA. Some distort the double helix by introducing sharp kinks. This can serve to relieve strain in tightly-bent DNA structures. Here, we design and test artificial architectural proteins based on a sequence-specific Transcription Activator-like Effector (TALE) protein, either alone or fused to a eukaryotic high mobility group B (HMGB) DNA-bending domain. We hypothesized that TALE protein binding would stiffen DNA to bending and twisting, acting as an architectural protein that antagonizes the formation of small DNA loops. In contrast, fusion to an HMGB domain was hypothesized to generate a targeted DNA-bending architectural protein that facilitates DNA looping. We provide evidence from Escherichia coli Lac repressor gene regulatory loops supporting these hypotheses in living bacteria. Both data fitting to a thermodynamic DNA looping model and sophisticated molecular modeling support the interpretation of these results. We find that TALE protein binding inhibits looping by stiffening DNA to bending and twisting, while the Nhp6A domain enhances looping by bending DNA without introducing twisting flexibility. Our work illustrates artificial approaches to sculpt DNA geometry with functional consequences. Similar approaches may be applicable to tune the stability of small DNA loops in eukaryotes.
Subject(s)
DNA, Bacterial/metabolism , DNA-Binding Proteins/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Lac Operon , Nucleic Acid Conformation , Promoter Regions, Genetic , Protein BindingABSTRACT
The quantitative polymerase chain reaction (qPCR) with detection of duplex DNA yield by intercalator fluorescence is a common and essential technique in nucleic acid analysis. We encountered unexpected results when applying standard qPCR methods to the quantitation of random DNA libraries flanked by regions of fixed sequence, a configuration essential for in vitro selection experiments. Here we describe the results of experiments revealing why conventional qPCR methods can fail to allow automated analysis in such cases, and simple solutions to this problem. In particular we show that renaturation of PCR products containing random regions is incomplete in late PCR cycles when extension fails due to reagent depletion. Intercalator fluorescence can then be lost at standard interrogation temperatures. We show that qPCR analysis of random DNA libraries can be achieved simply by adjusting the step at which intercalator fluorescence is monitored so that the yield of annealed constant regions is detected rather than the yield of full duplex DNA products.
Subject(s)
Aptamers, Nucleotide , Aptamers, Nucleotide/genetics , DNA/analysis , DNA/genetics , Gene Library , Intercalating Agents , Polymerase Chain Reaction/methodsABSTRACT
The yeast Nhp6A protein (yNhp6A) is a member of the eukaryotic HMGB family of chromatin factors that enhance apparent DNA flexibility. yNhp6A binds DNA nonspecifically with nM affinity, sharply bending DNA by >60°. It is not known whether the protein binds to unbent DNA and then deforms it, or if bent DNA conformations are 'captured' by protein binding. The former mechanism would be supported by discovery of conditions where unbent DNA is bound by yNhp6A. Here, we employed an array of conformational probes (FRET, fluorescence anisotropy, and circular dichroism) to reveal solution conditions in which an 18-base-pair DNA oligomer indeed remains bound to yNhp6A while unbent. In 100 mM NaCl, yNhp6A-bound DNA unbends as the temperature is raised, with no significant dissociation of the complex detected up to â¼45°C. In 200 mM NaCl, DNA unbending in the intact yNhp6A complex is again detected up to â¼35°C. Microseconds-resolved laser temperature-jump perturbation of the yNhp6a-DNA complex revealed relaxation kinetics that yielded unimolecular DNA bending/unbending rates on timescales of 500 µs-1 ms. These data provide the first direct observation of bending/unbending dynamics of DNA in complex with yNhp6A, suggesting a bind-then-bend mechanism for this protein.
Subject(s)
DNA, Fungal/chemistry , DNA, Fungal/metabolism , HMGN Proteins/chemistry , HMGN Proteins/metabolism , Nucleic Acid Conformation , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Chromatin Assembly and Disassembly/genetics , Fluorescence Resonance Energy Transfer , HMGN Proteins/physiology , Models, Molecular , Molecular Dynamics Simulation , Protein Binding , Protein Structure, Quaternary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/physiologyABSTRACT
Gene regulation by control of transcription initiation is a fundamental property of living cells. Much of our understanding of gene repression originated from studies of the Escherichia coli lac operon switch, in which DNA looping plays an essential role. To validate and generalize principles from lac for practical applications, we previously described artificial DNA looping driven by designed transcription activator-like effector dimer (TALED) proteins. Because TALE monomers bind the idealized symmetrical lac operator sequence in two orientations, our prior studies detected repression due to multiple DNA loops. We now quantitatively characterize gene repression in living E. coli by a collection of individual TALED loops with systematic loop length variation. Fitting of a thermodynamic model allows unequivocal demonstration of looping and comparison of the engineered TALED repression system with the natural lac repressor system.
Subject(s)
Escherichia coli Proteins , Transcription Activator-Like Effectors , DNA, Bacterial , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , Lac Operon/genetics , Lac Repressors/genetics , Lac Repressors/metabolism , Nucleic Acid ConformationABSTRACT
Genetic switches must alternate between states whose probabilities are dependent on regulatory signals. Classical examples of transcriptional control in bacteria depend on repressive DNA loops anchored by proteins whose structures are sensitive to small molecule inducers or co-repressors. We are interested in exploiting these natural principles to engineer artificial switches for transcriptional control of bacterial genes. Here, we implement designed homodimeric DNA looping proteins ('Transcription Activator-Like Effector Dimers'; TALEDs) for this purpose in living bacteria. Using well-studied FKBP dimerization domains, we build switches that mimic regulatory characteristics of classical Escherichia coli lactose, galactose and tryptophan operon promoters, including induction or co-repression by small molecules. Engineered DNA looping using TALEDs is thus a new approach to tuning gene expression in bacteria. Similar principles may also be applicable for gene control in eukaryotes.
Subject(s)
Gene Expression Regulation, Bacterial , Transcription Activator-Like Effectors/genetics , DNA/chemistry , Escherichia coli Proteins/metabolism , Lac Operon , Lac Repressors/metabolism , Models, Genetic , Protein Engineering , Protein Multimerization , Transcription Activator-Like Effectors/chemistry , Transcription Activator-Like Effectors/metabolismABSTRACT
Double-stranded DNA is a locally inflexible polymer that resists bending and twisting over hundreds of base pairs. Despite this, tight DNA bending is biologically important for DNA packaging in eukaryotic chromatin and tight DNA looping is important for gene repression in prokaryotes. We and others have previously shown that sequence nonspecific DNA kinking proteins, such as Escherichia coli heat unstable and Saccharomyces cerevisiae non-histone chromosomal protein 6A (Nhp6A), facilitate lac repressor (LacI) repression loops in E. coli. It has been unknown if this facilitation involves direct protein binding to the tightly bent DNA loop or an indirect effect promoting global negative supercoiling of DNA. Here we adapt two high-resolution in vivo protein-mapping techniques to demonstrate direct binding of the heterologous Nhp6A protein at a LacI repression loop in living E. coli cells.
Subject(s)
DNA, Bacterial/metabolism , Escherichia coli/genetics , HMGN Proteins/metabolism , Lac Repressors/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Chromatin Immunoprecipitation , DNA, Bacterial/chemistry , HMGN Proteins/chemistry , Lac Repressors/genetics , Promoter Regions, Genetic , Saccharomyces cerevisiae Proteins/chemistryABSTRACT
The Escherichia coli lactose operon provides a paradigm for understanding gene control by DNA looping where the lac repressor (LacI) protein competes with RNA polymerase for DNA binding. Not all promoter loops involve direct competition between repressor and RNA polymerase. This raises the possibility that positioning a promoter within a tightly constrained DNA loop is repressive per se, an idea that has previously only been considered in vitro. Here, we engineer living E. coli bacteria to measure repression due to promoter positioning within such a tightly constrained DNA loop in the absence of protein-protein binding competition. We show that promoters held within such DNA loops are repressed â¼100-fold, with up to an additional â¼10-fold repression (â¼1000-fold total) dependent on topological positioning of the promoter on the inner or outer face of the DNA loop. Chromatin immunoprecipitation data suggest that repression involves inhibition of both RNA polymerase initiation and elongation. These in vivo results show that gene repression can result from tightly looping promoter DNA even in the absence of direct competition between repressor and RNA polymerase binding.
Subject(s)
Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Lac Operon , Promoter Regions, Genetic , Binding, Competitive , DNA, Bacterial/genetics , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Silencing , Genes, Reporter , Lac Repressors/genetics , Lac Repressors/metabolism , Models, Genetic , Models, Molecular , Nucleic Acid Conformation , Protein Binding , beta-Galactosidase/biosynthesis , beta-Galactosidase/geneticsABSTRACT
The Escherichia coli lactose (lac) operon encodes the first genetic switch to be discovered, and lac remains a paradigm for studying negative and positive control of gene expression. Negative control is believed to involve competition of RNA polymerase and Lac repressor for overlapping binding sites. Contributions to the local Lac repressor concentration come from free repressor and repressor delivered to the operator from remote auxiliary operators by DNA looping. Long-standing questions persist concerning the actual role of DNA looping in the mechanism of promoter repression. Here, we use experiments in living bacteria to resolve four of these questions. We show that the distance dependence of repression enhancement is comparable for upstream and downstream auxiliary operators, confirming the hypothesis that repressor concentration increase is the principal mechanism of repression loops. We find that as few as four turns of DNA can be constrained in a stable loop by Lac repressor. We show that RNA polymerase is not trapped at repressed promoters. Finally, we show that constraining a promoter in a tight DNA loop is sufficient for repression even when promoter and operator do not overlap.
Subject(s)
DNA, Bacterial/chemistry , Gene Expression Regulation, Bacterial , Lac Repressors/metabolism , Promoter Regions, Genetic , Transcription, Genetic , DNA, Bacterial/metabolism , DNA-Directed RNA Polymerases/metabolism , Models, Genetic , Operator Regions, GeneticABSTRACT
LacI/GalR transcription regulators have extensive, non-conserved interfaces between their regulatory domains and the 18 amino acids that serve as 'linkers' to their DNA-binding domains. These non-conserved interfaces might contribute to functional differences between paralogs. Previously, two chimeras created by domain recombination displayed novel functional properties. Here, we present a synthetic protein family, which was created by joining the LacI DNA-binding domain/linker to seven additional regulatory domains. Despite 'mismatched' interfaces, chimeras maintained allosteric response to their cognate effectors. Therefore, allostery in many LacI/GalR proteins does not require interfaces with precisely matched interactions. Nevertheless, the chimeric interfaces were not silent to mutagenesis, and preliminary comparisons suggest that the chimeras provide an ideal context for systematically exploring functional contributions of non-conserved positions. DNA looping experiments revealed higher order (dimer-dimer) oligomerization in several chimeras, which might be possible for the natural paralogs. Finally, the biological significance of repression differences was determined by measuring bacterial growth rates on lactose minimal media. Unexpectedly, moderate and strong repressors showed an apparent induction phase, even though inducers were not provided; therefore, an unknown mechanism might contribute to regulation of the lac operon. Nevertheless, altered growth correlated with altered repression, which indicates that observed functional modifications are significant.
Subject(s)
Escherichia coli Proteins/chemistry , Gene Expression Regulation, Bacterial , Lac Repressors/chemistry , Repressor Proteins/chemistry , Transcription, Genetic , Allosteric Regulation , DNA, Bacterial/chemistry , DNA, Bacterial/metabolism , Escherichia coli/genetics , Escherichia coli/growth & development , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Lac Operon , Lac Repressors/genetics , Lac Repressors/metabolism , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Up-RegulationABSTRACT
The occurrence of DNA looping is ubiquitous. This process plays a well-documented role in the regulation of prokaryotic gene expression, such as in regulation of the Escherichia coli lactose (lac) operon. Here we present two complementary methods for high-resolution in vivo detection of DNA/protein binding within the bacterial nucleoid by using either chromatin immunoprecipitation combined with phage λ exonuclease digestion (ChIP-exo) or chromatin endogenous cleavage (ChEC), coupled with ligation-mediated polymerase chain reaction (LM-PCR) and Southern blot analysis. As an example, we apply these in vivo protein-mapping methods to E. coli to show direct binding of architectural proteins in the Lac repressor-mediated DNA repression loop.
Subject(s)
Chromatin Immunoprecipitation , DNA, Bacterial , Escherichia coli , DNA, Bacterial/metabolism , DNA, Bacterial/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Chromatin Immunoprecipitation/methods , Protein Binding , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Lac Operon , Polymerase Chain Reaction/methods , Blotting, Southern , Bacteriophage lambda/genetics , Bacteriophage lambda/metabolismABSTRACT
Whether TMPRSS2-ERG fusion and TP53 gene alteration coordinately promote prostate cancer (PCa) remains unclear. Here we demonstrate that TMPRSS2-ERG fusion and TP53 mutation / deletion co-occur in PCa patient specimens and this co-occurrence accelerates prostatic oncogenesis. p53 gain-of-function (GOF) mutants are now shown to bind to a unique DNA sequence in the CTNNB1 gene promoter and transactivate its expression. ERG and ß-Catenin co-occupy sites at pyrimidine synthesis gene (PSG) loci and promote PSG expression, pyrimidine synthesis and PCa growth. ß-Catenin inhibition by small molecule inhibitors or oligonucleotide-based PROTAC suppresses TMPRSS2-ERG- and p53 mutant-positive PCa cell growth in vitro and in mice. Our study identifies a gene transactivation function of GOF mutant p53 and reveals ß-Catenin as a transcriptional target gene of p53 GOF mutants and a driver and therapeutic target of TMPRSS2-ERG- and p53 GOF mutant-positive PCa.
Subject(s)
Prostatic Neoplasms , Transcriptional Regulator ERG , Tumor Suppressor Protein p53 , Animals , Humans , Male , Mice , beta Catenin/genetics , beta Catenin/metabolism , Gain of Function Mutation , Oncogene Proteins, Fusion/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Proto-Oncogenes , Pyrimidines/biosynthesis , Transcriptional Regulator ERG/genetics , Transcriptional Regulator ERG/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
The inflexibility of double-stranded DNA with respect to bending and twisting is well established in vitro. Understanding apparent DNA physical properties in vivo is a greater challenge. Here, we exploit repression looping with components of the Escherichia coli lac operon to monitor DNA flexibility in living cells. We create a minimal system for testing the shortest possible DNA repression loops that contain an E. coli promoter, and compare the results to prior experiments. Our data reveal that loop-independent repression occurs for certain tight operator/promoter spacings. When only loop-dependent repression is considered, fits to a thermodynamic model show that DNA twisting limits looping in vivo, although the apparent DNA twist flexibility is 2- to 4-fold higher than in vitro. In contrast, length-dependent resistance to DNA bending is not observed in these experiments, even for the shortest loops constraining <0.4 persistence lengths of DNA. As observed previously for other looping configurations, loss of the nucleoid protein heat unstable (HU) markedly disables DNA looping in vivo. Length-independent DNA bending energy may reflect the activities of architectural proteins and the structure of the DNA topological domain. We suggest that the shortest loops are formed in apical loops rather than along the DNA plectonemic superhelix.
Subject(s)
DNA, Bacterial/chemistry , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Lac Operon , DNA, Bacterial/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Escherichia coli Proteins/genetics , Lac Repressors/metabolism , Nucleic Acid Conformation , Operator Regions, Genetic , Promoter Regions, GeneticABSTRACT
DNA looping is important for gene repression and activation in Escherichia coli and is necessary for some kinds of gene regulation and recombination in eukaryotes. We are interested in sequence-nonspecific architectural DNA-binding proteins that alter the apparent flexibility of DNA by producing transient bends or kinks in DNA. The bacterial heat unstable (HU) and eukaryotic high-mobility group B (HMGB) proteins fall into this category. We have exploited a sensitive genetic assay of DNA looping in living E. coli cells to explore the extent to which HMGB proteins and derivatives can complement a DNA looping defect in E. coli lacking HU protein. Here, we show that derivatives of the yeast HMGB protein Nhp6A rescue DNA looping in E. coli lacking HU, in some cases facilitating looping to a greater extent than is observed in E. coli expressing normal levels of HU protein. Nhp6A-induced changes in the DNA length-dependence of repression efficiency suggest that Nhp6A alters DNA twist in vivo. In contrast, human HMGB2-box A derivatives did not rescue looping.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Escherichia coli Proteins/genetics , HMGB Proteins/chemistry , Nuclear Proteins/chemistry , Repressor Proteins/genetics , Saccharomyces cerevisiae Proteins/chemistry , Amino Acid Sequence , Base Sequence , DNA/chemistry , Escherichia coli/genetics , Gene Deletion , Gene Expression Regulation, Bacterial , Genetic Complementation Test , HMGB Proteins/genetics , HMGB Proteins/metabolism , HMGB2 Protein/chemistry , HMGB2 Protein/genetics , HMGN Proteins , Humans , Molecular Sequence Data , Nuclear Proteins/genetics , Operator Regions, Genetic , Phenotype , Promoter Regions, Genetic , Protein Structure, Tertiary , Saccharomyces cerevisiae Proteins/genetics , Sequence DeletionABSTRACT
HMGB proteins are abundant non-histone components of eukaryotic chromatin. The biological function of DNA sequence-nonspecific HMGB proteins is obscure. These proteins are composed of one or two conserved HMG box domains, each forming three alpha-helices that fold into a sequence-nonspecific DNA-binding module recognizing the DNA minor groove. Box A and box B homology domains have subtle sequence differences such that box B domains bend DNA strongly while DNA bending by isolated box A domains is weaker. Both box A and box B domains preferentially bind to distorted DNA structures. Here we show using DNA cyclization kinetics assays in vitro and Escherichia coli DNA looping assays in vivo that an isolated HMG box A domain derived from human HMGB2 folds poorly and does not enhance apparent DNA flexibility. Surprisingly, substitution of a small number of cationic residues from the N-terminal leader of a functional yeast box B protein, Nhp6Ap, confers the ability to enhance DNA flexibility. These results demonstrate important roles for cationic leader amino acids in HMGB folding, DNA interaction, and DNA bending.
Subject(s)
DNA/chemistry , HMG-Box Domains/physiology , HMGB Proteins/chemistry , Nucleic Acid Conformation , Circular Dichroism , DNA/metabolism , DNA-Binding Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Fluorescence Polarization , HMGB Proteins/genetics , HMGB Proteins/metabolism , HMGB2 Protein/chemistry , HMGB2 Protein/genetics , HMGB2 Protein/metabolism , HMGN Proteins/chemistry , HMGN Proteins/genetics , HMGN Proteins/metabolism , Humans , Lac Operon/genetics , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Binding/genetics , Protein Sorting Signals/genetics , Protein Structure, Secondary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
The intrinsic stiffness of DNA limits its ability to be bent and twisted over short lengths, but such deformations are required for gene regulation. One classic paradigm is DNA looping in the regulation of the Escherichia coli lac operon. Lac repressor protein binds simultaneously to two operator sequences flanking the lac promoter. Analysis of the length dependence of looping-dependent repression of the lac operon provides insight into DNA deformation energetics within cells. The apparent flexibility of DNA is greater in vivo than in vitro, possibly because of host proteins that bind DNA and induce sites of flexure. Here we test DNA looping in bacterial strains lacking the nucleoid proteins HU, IHF or H-NS. We confirm that deletion of HU inhibits looping and that quantitative modeling suggests residual looping in the induced operon. Deletion of IHF has little effect. Remarkably, DNA looping is strongly enhanced in the absence of H-NS, and an explanatory model is proposed. Chloroquine titration, psoralen crosslinking and supercoiling-sensitive reporter assays show that the effects of nucleoid proteins on looping are not correlated with their effects on either total or unrestrained supercoiling. These results suggest that host nucleoid proteins can directly facilitate or inhibit DNA looping in bacteria.
Subject(s)
DNA, Bacterial/chemistry , DNA-Binding Proteins/physiology , Escherichia coli Proteins/physiology , Escherichia coli/genetics , Lac Operon , Bacterial Proteins/genetics , Bacterial Proteins/physiology , DNA, Superhelical/chemistry , DNA-Binding Proteins/genetics , Escherichia coli Proteins/genetics , Gene Deletion , Integration Host Factors/genetics , Integration Host Factors/physiology , Models, Genetic , Nucleic Acid ConformationABSTRACT
Repression of a promoter by entrapment within a tightly bent DNA loop is a common mechanism of gene regulation in bacteria. Besides the mechanical properties of the looped DNA and affinity of the protein that anchors the loop, cellular energetics and DNA negative supercoiling are likely factors determining the stability of the repression loop. E. coli cells undergo numerous highly regulated and dynamic transitions as resources are depleted during bacterial growth. We hypothesized that the probability of DNA looping depends on the growth status of the E. coli culture. We utilized a well-characterized repression loop model assembled from elements of the lac operon to measure loop length-dependent repression at three different culture densities. Remarkably, even with changes in supercoiling, there exists a dynamic compensation in which the contribution of DNA looping to gene repression remains essentially constant.
ABSTRACT
Immobilization of divalent Nickel cations provides a tool for affinity purification of proteins containing hexahistidine tags. During experiments to generate single-stranded DNA aptamers to immobilized proteins we inadvertently identified DNA sequences with affinity for Nickel-nitrilotriacetic acid (Ni(2+)-NTA) magnetic beads. Analysis of these aptamers revealed that affinity for the Ni(2+)-NTA support requires only single-stranded sequences with multiple adenosine residues. Bound nucleic acids can be eluted with imidazole. A single-stranded dA(20) affinity tag (but not other homopolymer sequences) is sufficient for immobilization of double-stranded DNA PCR products on Ni(2+)-NTA magnetic beads. Addition of an rA(20) sequence to an RNA transcript allowed its affinity capture on Ni(2+)-NTA magnetic beads, suggesting an approach for purification of poly(A) mRNA.
Subject(s)
DNA/chemistry , Nickel/chemistry , RNA/chemistry , Sequence Analysis, DNA/methods , Base Sequence , Binding Sites , Molecular Sequence DataABSTRACT
The occurrence of DNA looping is ubiquitous. This process plays a well-documented role in the regulation of prokaryotic gene expression, such as the Escherichia coli lactose (lac) operon. Here, we present two complementary methods for high-resolution in vivo detection of DNA/protein binding within the bacterial nucleoid by using either chromatin immunoprecipitation combined with phage λ exonuclease digestion (ChIP-exo) or chromatin endogenous cleavage (ChEC), coupled with ligation-mediated polymerase chain reaction (LM-PCR) and Southern blot analysis. As an example we apply these in vivo protein-mapping methods to E. coli to show direct binding of architectural proteins in the Lac repressor-mediated DNA repression loop.
Subject(s)
Bacteria/genetics , Bacteria/metabolism , Chromatin Immunoprecipitation , DNA, Bacterial/metabolism , DNA-Binding Proteins/metabolism , Blotting, Southern , Chromatin Immunoprecipitation/methods , DNA Cleavage , Exonucleases/metabolism , Polymerase Chain ReactionABSTRACT
The Escherichia coli lac operon provides a classic paradigm for understanding regulation of gene transcription. It is now appreciated that lac promoter repression involves cooperative binding of the bidentate lac repressor tetramer to pairs of lac operators via DNA looping. We have adapted components of this system to create an artificial assay of DNA flexibility in E.coli. This approach allows for systematic study of endogenous and exogenous proteins as architectural factors that enhance apparent DNA flexibility in vivo. We show that inducer binding does not completely remove repression loops but it does alter their geometries. Deletion of the E.coli HU protein drastically destabilizes small repression loops, an effect that can be partially overcome by expression of a heterologous mammalian HMG protein. These results emphasize that the inherent torsional inflexibility of DNA restrains looping and must be modulated in vivo.