RESUMEN
Genes are often transcribed by multiple RNA polymerases (RNAPs) at densities that can vary widely across genes and environmental conditions. Here, we provide in vitro and in vivo evidence for a built-in mechanism by which co-transcribing RNAPs display either collaborative or antagonistic dynamics over long distances (>2 kb) through transcription-induced DNA supercoiling. In Escherichia coli, when the promoter is active, co-transcribing RNAPs translocate faster than a single RNAP, but their average speed is not altered by large variations in promoter strength and thus RNAP density. Environmentally induced promoter repression reduces the elongation efficiency of already-loaded RNAPs, causing premature termination and quick synthesis arrest of no-longer-needed proteins. This negative effect appears independent of RNAP convoy formation and is abrogated by topoisomerase I activity. Antagonistic dynamics can also occur between RNAPs from divergently transcribed gene pairs. Our findings may be broadly applicable given that transcription on topologically constrained DNA is the norm across organisms.
Asunto(s)
ADN Bacteriano/genética , ADN Superhelicoidal/genética , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/genética , Transcripción Genética , ARN Polimerasas Dirigidas por ADN/química , Regulación Bacteriana de la Expresión Génica/genética , Glucosa/farmacología , Glicósidos/farmacología , Isopropil Tiogalactósido/farmacología , Cinética , Operón Lac/efectos de los fármacos , Operón Lac/genética , Plásmidos/genética , Regiones Promotoras Genéticas/genética , ARN Bacteriano/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Rifampin/farmacologíaRESUMEN
Proteins that are kinetically stable are thought to be less prone to both aggregation and proteolysis. We demonstrate that the classical lac system of Escherichia coli can be leveraged as a model system to study this relation. ß-galactosidase (LacZ) plays a critical role in lactose metabolism and is an extremely stable protein that can persist in growing cells for multiple generations after expression has stopped. By attaching degradation tags to the LacZ protein, we find that LacZ can be transiently degraded during lac operon expression but once expression has stopped functional LacZ is protected from degradation. We reversibly destabilize its tetrameric assembly using α-complementation, and show that unassembled LacZ monomers and dimers can either be degraded or lead to formation of aggregates within cells, while the tetrameric state protects against proteolysis and aggregation. We show that the presence of aggregates is associated with cell death, and that these proteotoxic stress phenotypes can be alleviated by attaching an ssrA tag to LacZ monomers which leads to their degradation. We unify our findings using a biophysical model that enables the interplay of protein assembly, degradation, and aggregation to be studied quantitatively in vivo. This work may yield approaches to reversing and preventing protein-misfolding disease states, while elucidating the functions of proteolytic stability in constant and fluctuating environments.
Asunto(s)
Escherichia coli , Operón Lac , Proteolisis , beta-Galactosidasa , beta-Galactosidasa/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Agregado de Proteínas , Estabilidad de EnzimasRESUMEN
Cellular regulation is believed to evolve in response to environmental variability. However, this has been difficult to test directly. Here, we show that a gene regulation system evolves to the optimal regulatory response when challenged with variable environments. We engineered a genetic module subject to regulation by the lac repressor (LacI) in E. coli, whose expression is beneficial in one environmental condition and detrimental in another. Measured tradeoffs in fitness between environments predict the competition between regulatory phenotypes. We show that regulatory evolution in adverse environments is delayed at specific boundaries in the phenotype space of the regulatory LacI protein. Once this constraint is relieved by mutation, adaptation proceeds toward the optimum, yielding LacI with an altered allosteric mechanism that enables an opposite response to its regulatory ligand IPTG. Our results indicate that regulatory evolution can be understood in terms of tradeoff optimization theory.
Asunto(s)
Evolución Biológica , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Regulación Alostérica , Proteínas de Escherichia coli/metabolismo , Aptitud Genética , Isopropil Tiogalactósido/metabolismo , Operón Lac , Represoras Lac/metabolismo , MutaciónRESUMEN
Optogenetics' advancement has made light induction attractive for controlling biological processes due to its advantages of fine-tunability, reversibility, and low toxicity. The lactose operon induction system, commonly used in Escherichia coli, relies on the binding of lactose or isopropyl ß-d-1-thiogalactopyranoside (IPTG) to the lactose repressor protein LacI, playing a pivotal role in controlling the lactose operon. Here, we harnessed the light-responsive light-oxygen-voltage 2 (LOV2) domain from Avena sativa phototropin 1 as a tool for light control and engineered LacI into two light-responsive variants, OptoLacIL and OptoLacID. These variants exhibit direct responsiveness to light and darkness, respectively, eliminating the need for IPTG. Building upon OptoLacI, we constructed two light-controlled E. coli gene expression systems, OptoE.coliLight system and OptoE.coliDark system. These systems enable bifunctional gene expression regulation in E. coli through light manipulation and show superior controllability compared to IPTG-induced systems. We applied the OptoE.coliDark system to protein production and metabolic flux control. Protein production levels are comparable to those induced by IPTG. Notably, the titers of dark-induced production of 1,3-propanediol (1,3-PDO) and ergothioneine exceeded 110% and 60% of those induced by IPTG, respectively. The development of OptoLacI will contribute to the advancement of the field of optogenetic protein engineering, holding substantial potential applications across various fields.
Asunto(s)
Escherichia coli , Isopropil Tiogalactósido , Operón Lac , Represoras Lac , Luz , Optogenética , Isopropil Tiogalactósido/farmacología , Represoras Lac/metabolismo , Represoras Lac/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/efectos de la radiación , Optogenética/métodos , Regulación Bacteriana de la Expresión Génica/efectos de la radiación , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ingeniería de Proteínas/métodos , Avena/genética , Avena/metabolismo , Avena/efectos de la radiaciónRESUMEN
High-resolution NMR spectroscopy enabled us to characterize allosteric transitions between various functional states of the dimeric Escherichia coli Lac repressor. In the absence of ligands, the dimer exists in a dynamic equilibrium between DNA-bound and inducer-bound conformations. Binding of either effector shifts this equilibrium toward either bound state. Analysis of the ternary complex between repressor, operator DNA, and inducer shows how adding the inducer results in allosteric changes that disrupt the interdomain contacts between the inducer binding and DNA binding domains and how this in turn leads to destabilization of the hinge helices and release of the Lac repressor from the operator. Based on our data, the allosteric mechanism of the induction process is in full agreement with the well-known Monod-Wyman-Changeux model.
Asunto(s)
Proteínas de Escherichia coli , Represoras Lac/genética , Represoras Lac/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulación Alostérica/genética , Escherichia coli/metabolismo , ADN/metabolismo , Estructura Secundaria de Proteína , Operón Lac/genéticaRESUMEN
DNA looping has emerged as a central paradigm of transcriptional regulation, as it is shared across many living systems. One core property of DNA looping-based regulation is its ability to greatly enhance repression or activation of genes with only a few copies of transcriptional regulators. However, this property based on a small number of proteins raises the question of the robustness of such a mechanism with respect to the large intracellular perturbations taking place during growth and division of the cell. Here we address the issue of sensitivity to variations of intracellular parameters of gene regulation by DNA looping. We use the lac system as a prototype to experimentally identify the key features of the robustness of DNA looping in growing Escherichia coli cells. Surprisingly, we observe time intervals of tight repression spanning across division events, which can sometimes exceed 10 generations. Remarkably, the distribution of such long time intervals exhibits memoryless statistics that is mostly insensitive to repressor concentration, cell division events, and the number of distinct loops accessible to the system. By contrast, gene regulation becomes highly sensitive to these perturbations when DNA looping is absent. Using stochastic simulations, we propose that the observed robustness to division emerges from the competition between fast, multiple rebinding events of repressors and slow initiation rate of the RNA polymerase. We argue that fast rebinding events are a direct consequence of DNA looping that ensures robust gene repression across a range of intracellular perturbations.
Asunto(s)
División Celular , ADN Bacteriano , Operón Lac , División Celular/genética , ADN Bacteriano/química , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Represoras Lac/genética , Represoras Lac/metabolismo , Conformación de Ácido Nucleico , Análisis de la Célula IndividualRESUMEN
Pasteurella multocida is a zoonotic conditional pathogen that infects multiple livestock species, causing substantial economic losses in the animal husbandry industry. An efficient markerless method for gene manipulation may facilitate the investigations of P. multocida gene function and pathogenesis of P. multocida. Herein, a temperature-sensitive shuttle vector was constructed using lacZ as a selection marker, and markerless glgB, opa, and hyaE mutants of P. multocida were subsequently constructed through blue-white colony screening. The screening efficiency of markerless deletion strains was improved by the lacZ system, and the method could be used for multiple gene deletions. However, the fur mutant was unavailable via this method. Therefore, we constructed a pheSm screening system based on mutated phenylalanine tRNA synthetase as a counterselection marker to achieve fur deletion mutant. The transformed strain was sensitive to 20 mM p-chloro-phenylalanine, demonstrating the feasibility of pheSm as a counter-selective marker. The pheSm system was used for markerless deletions of glgB, opa, and hyaE as well as fur that could not be screened by the lacZ system. A comparison of screening efficiencies of the system showed that the pheSm counterselection system was more efficient than the lacZ system and broadly applicable for mutant screening. The methods developed herein may provide valuable tools for genetic manipulation of P. multocida.IMPORTANCEPasteurella multocida is a highly contagious zoonotic pathogen. An understanding of its underlying pathogenic mechanisms is of considerable importance and requires efficient species-specific genetic tools. Herein, we propose a screening system for P. multocida mutants using lacZ or pheSm screening markers. We evaluated the efficiencies of both systems, which were used to achieve markerless deletion of multiple genes. The results of this study support the use of lacZ or pheSm as counterselection markers to improve counterselection efficiency in P. multocida. This study provides an effective genetic tool for investigations of the virulence gene functions and pathogenic mechanisms of P. multocida.
Asunto(s)
Pasteurella multocida , Animales , Pasteurella multocida/genética , Operón Lac , Vectores Genéticos , FenilalaninaRESUMEN
Detailed genomic contact maps have revealed that chromosomes are structurally organized in megabase-sized topologically associated domains (TADs) that encompass smaller subTADs. These domains segregate in the nuclear space to form active and inactive nuclear compartments, but cause and consequence of compartmentalization are largely unknown. Here, we combined lacO/lacR binding platforms with allele-specific 4C technologies to track their precise position in the three-dimensional genome upon recruitment of NANOG, SUV39H1, or EZH2. We observed locked genomic loci resistant to spatial repositioning and unlocked loci that could be repositioned to different nuclear subcompartments with distinct chromatin signatures. Focal protein recruitment caused the entire subTAD, but not surrounding regions, to engage in new genomic contacts. Compartment switching was found uncoupled from transcription changes, and the enzymatic modification of histones per se was insufficient for repositioning. Collectively, this suggests that trans-associated factors influence three-dimensional compartmentalization independent of their cis effect on local chromatin composition and activity.
Asunto(s)
Núcleo Celular/metabolismo , Segregación Cromosómica , Células Madre Embrionarias/metabolismo , Sitios Genéticos , Operón Lac , Represoras Lac/metabolismo , Animales , Células Cultivadas , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Proteína Potenciadora del Homólogo Zeste 2 , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Represoras Lac/genética , Metiltransferasas/genética , Metiltransferasas/metabolismo , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Proteína Homeótica Nanog , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , TransfecciónRESUMEN
In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause ("initiation pause," â¼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , ARN Mensajero/genética , Transcripción Genética , Secuencia de Bases , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Operón Lac , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Regiones Promotoras Genéticas , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , Rifampin/farmacología , Imagen Individual de Molécula/métodos , Factores de TiempoRESUMEN
Some proteins, like the lac repressor (LacI), mediate long-range loops that alter DNA topology and create torsional barriers. During transcription, RNA polymerase generates supercoiling that may facilitate passage through such barriers. We monitored E. coli RNA polymerase progress along templates in conditions that prevented, or favored, 400 bp LacI-mediated DNA looping. Tethered particle motion measurements revealed that RNA polymerase paused longer at unlooped LacI obstacles or those barring entry to a loop than those barring exit from the loop. Enhanced dissociation of a LacI roadblock by the positive supercoiling generated ahead of a transcribing RNA polymerase within a torsion-constrained DNA loop may be responsible for this reduction in pause time. In support of this idea, RNA polymerase transcribed 6-fold more slowly through looped DNA and paused at LacI obstacles for 66% less time on positively supercoiled compared to relaxed templates, especially under increased tension (torque). Positive supercoiling propagating ahead of polymerase facilitated elongation along topologically complex, protein-coated templates.
Asunto(s)
ADN , Escherichia coli , ADN/genética , ADN/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ADN Superhelicoidal/genética , ADN Superhelicoidal/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Operón Lac , Represoras Lac/genética , Represoras Lac/metabolismo , Conformación de Ácido NucleicoRESUMEN
Notch signaling controls many developmental processes by regulating gene expression. Notch-dependent enhancers recruit activation complexes consisting of the Notch intracellular domain, the Cbf/Su(H)/Lag1 (CSL) transcription factor (TF), and the Mastermind co-factor via two types of DNA sites: monomeric CSL sites and cooperative dimer sites called Su(H) paired sites (SPS). Intriguingly, the CSL TF can also bind co-repressors to negatively regulate transcription via these same sites. Here, we tested how synthetic enhancers with monomeric CSL sites versus dimeric SPSs bind Drosophila Su(H) complexes in vitro and mediate transcriptional outcomes in vivo. Our findings reveal that while the Su(H)/Hairless co-repressor complex similarly binds SPS and CSL sites in an additive manner, the Notch activation complex binds SPSs, but not CSL sites, in a cooperative manner. Moreover, transgenic reporters with SPSs mediate stronger, more consistent transcription and are more resistant to increased Hairless co-repressor expression compared to reporters with the same number of CSL sites. These findings support a model in which SPS containing enhancers preferentially recruit cooperative Notch activation complexes over Hairless repression complexes to ensure consistent target gene activation.
Asunto(s)
Proteínas de Drosophila/fisiología , Elementos de Facilitación Genéticos , Receptores Notch/metabolismo , Proteínas Represoras/fisiología , Factores de Transcripción/fisiología , Animales , Sitios de Unión , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulación de la Expresión Génica , Genes Reporteros , Operón Lac , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Activación TranscripcionalRESUMEN
Vibrio cholerae is the causative agent of cholera, a notorious diarrheal disease that is typically transmitted via contaminated drinking water. The current pandemic agent, the El Tor biotype, has undergone several genetic changes that include horizontal acquisition of two genomic islands (VSP-I and VSP-II). VSP presence strongly correlates with pandemicity; however, the contribution of these islands to V. cholerae's life cycle, particularly the 26-kb VSP-II, remains poorly understood. VSP-II-encoded genes are not expressed under standard laboratory conditions, suggesting that their induction requires an unknown signal from the host or environment. One signal that bacteria encounter under both host and environmental conditions is metal limitation. While studying V. cholerae's zinc-starvation response in vitro, we noticed that a mutant constitutively expressing zinc starvation genes (Δzur) congregates at the bottom of a culture tube when grown in a nutrient-poor medium. Using transposon mutagenesis, we found that flagellar motility, chemotaxis, and VSP-II encoded genes were required for congregation. The VSP-II genes encode an AraC-like transcriptional activator (VerA) and a methyl-accepting chemotaxis protein (AerB). Using RNA-seq and lacZ transcriptional reporters, we show that VerA is a novel Zur target and an activator of the nearby AerB chemoreceptor. AerB interfaces with the chemotaxis system to drive oxygen-dependent congregation and energy taxis. Importantly, this work suggests a functional link between VSP-II, zinc-starved environments, and energy taxis, yielding insights into the role of VSP-II in a metal-limited host or aquatic reservoir.
Asunto(s)
Quimiotaxis/genética , Regulación Bacteriana de la Expresión Génica , Islas Genómicas , Proteínas Represoras/genética , Vibrio cholerae/genética , Vibrio cholerae/patogenicidad , Zinc/deficiencia , Adhesión Bacteriana , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cólera/microbiología , Cólera/patología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genes Reporteros , Genoma Bacteriano , Humanos , Operón Lac , Oxígeno/metabolismo , Oxígeno/farmacología , Pandemias , Proteínas Represoras/metabolismo , Transcripción Genética , Vibrio cholerae/efectos de los fármacos , Vibrio cholerae/metabolismo , Zinc/farmacologíaRESUMEN
Control of the lac operon with isopropyl ß-D-1-thiogalactopyranoside (IPTG) has been used to regulate gene expression in Escherichia coli for countless applications, including metabolic engineering and recombinant protein production. However, optogenetics offers unique capabilities, such as easy tunability, reversibility, dynamic induction strength and spatial control, that are difficult to obtain with chemical inducers. We have developed a series of circuits for optogenetic regulation of the lac operon, which we call OptoLAC, to control gene expression from various IPTG-inducible promoters using only blue light. Applying them to metabolic engineering improves mevalonate and isobutanol production by 24% and 27% respectively, compared to IPTG induction, in light-controlled fermentations scalable to at least two-litre bioreactors. Furthermore, OptoLAC circuits enable control of recombinant protein production, reaching yields comparable to IPTG induction but with easier tunability of expression. OptoLAC circuits are potentially useful to confer light control over other cell functions originally designed to be IPTG-inducible.
Asunto(s)
Escherichia coli/efectos de la radiación , Regulación Bacteriana de la Expresión Génica , Operón Lac/efectos de la radiación , Ingeniería Metabólica/métodos , Optogenética/métodos , Reactores Biológicos , Butanoles/metabolismo , Butanoles/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Isopropil Tiogalactósido/farmacología , Luz , Fototransducción , Ácido Mevalónico/metabolismo , Ácido Mevalónico/farmacología , Regiones Promotoras GenéticasRESUMEN
Populations of bacteria often undergo a lag in growth when switching conditions. Because growth lags can be large compared to typical doubling times, variations in growth lag are an important but often overlooked component of bacterial fitness in fluctuating environments. We here explore how growth lag variation is determined for the archetypical switch from glucose to lactose as a carbon source in Escherichia coli. First, we show that single-cell lags are bimodally distributed and controlled by a single-molecule trigger. That is, gene expression noise causes the population before the switch to divide into subpopulations with zero and nonzero lac operon expression. While "sensorless" cells with zero preexisting lac expression at the switch have long lags because they are unable to sense the lactose signal, any nonzero lac operon expression suffices to ensure a short lag. Second, we show that the growth lag at the population level depends crucially on the fraction of sensorless cells and that this fraction in turn depends sensitively on the growth condition before the switch. Consequently, even small changes in basal expression can significantly affect the fraction of sensorless cells, thereby population lags and fitness under switching conditions, and may thus be subject to significant natural selection. Indeed, we show that condition-dependent population lags vary across wild E. coli isolates. Since many sensory genes are naturally low expressed in conditions where their inducer is not present, bimodal responses due to subpopulations of sensorless cells may be a general mechanism inducing phenotypic heterogeneity and controlling population lags in switching environments. This mechanism also illustrates how gene expression noise can turn even a simple sensory gene circuit into a bet hedging module and underlines the profound role of gene expression noise in regulatory responses.
Asunto(s)
Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Aptitud Genética/fisiología , Bacterias/genética , Bacterias/metabolismo , Ambiente , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Redes Reguladoras de Genes/genética , Interacción Gen-Ambiente , Aptitud Genética/genética , Glucosa/metabolismo , Operón Lac , Lactosa/metabolismo , FenotipoRESUMEN
Mutagenicity testing is an essential component of health safety assessment. Duplex Sequencing (DS), an emerging high-accuracy DNA sequencing technology, may provide substantial advantages over conventional mutagenicity assays. DS could be used to eliminate reliance on standalone reporter assays and provide mechanistic information alongside mutation frequency (MF) data. However, the performance of DS must be thoroughly assessed before it can be routinely implemented for standard testing. We used DS to study spontaneous and procarbazine (PRC)-induced mutations in the bone marrow (BM) of MutaMouse males across a panel of 20 diverse genomic targets. Mice were exposed to 0, 6.25, 12.5, or 25 mg/kg-bw/day for 28 days by oral gavage and BM sampled 42 days post-exposure. Results were compared with those obtained using the conventional lacZ viral plaque assay on the same samples. DS detected significant increases in mutation frequencies and changes to mutation spectra at all PRC doses. Low intra-group variability within DS samples allowed for detection of increases at lower doses than the lacZ assay. While the lacZ assay initially yielded a higher fold-change in mutant frequency than DS, inclusion of clonal mutations in DS mutation frequencies reduced this discrepancy. Power analyses suggested that three animals per dose group and 500 million duplex base pairs per sample is sufficient to detect a 1.5-fold increase in mutations with > 80% power. Overall, we demonstrate several advantages of DS over classical mutagenicity assays and provide data to support efforts to identify optimal study designs for the application of DS as a regulatory test.
Asunto(s)
Médula Ósea , Tasa de Mutación , Masculino , Ratones , Animales , Procarbazina/toxicidad , Mutágenos/toxicidad , Mutación , Pruebas de Mutagenicidad/métodos , Ratones Transgénicos , Operón LacRESUMEN
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.
Asunto(s)
ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Operón Lac , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
Proteins that can bring together separate DNA sites, either on the same or on different DNA molecules, are critical for a variety of DNA-based processes. However, there are no general and technically simple assays to detect proteins capable of DNA looping in vivo nor to quantitate their in vivo looping efficiency. Here, we develop a quantitative in vivo assay for DNA-looping proteins in Escherichia coli that requires only basic DNA cloning techniques and a LacZ assay. The assay is based on loop assistance, where two binding sites for the candidate looping protein are inserted internally to a pair of operators for the E. coli LacI repressor. DNA looping between the sites shortens the effective distance between the lac operators, increasing LacI looping and strengthening its repression of a lacZ reporter gene. Analysis based on a general model for loop assistance enables quantitation of the strength of looping conferred by the protein and its binding sites. We use this 'loopometer' assay to measure DNA looping for a variety of bacterial and phage proteins.
Asunto(s)
Técnicas de Química Analítica , ADN Bacteriano/genética , Proteínas de Escherichia coli/química , Escherichia coli/genética , Operón Lac , Represoras Lac/química , Bacteriófago lambda/genética , Sitios de Unión , Proteínas de Escherichia coli/genética , Represoras Lac/genética , Conformación de Ácido Nucleico , Regiones Operadoras Genéticas , Unión Proteica , Proteínas Represoras/genética , Proteínas Reguladoras y Accesorias Virales/genéticaRESUMEN
Laccase genes produce laccase enzymes that play a crucial role in the production of lignin and oxidation reactions within plants. Lignin is a complex polymer that provides structure and toughness to the cell walls of numerous fruit plants. The LAC genes that encode laccase enzymes play vital roles in plant physiology, including the synthesis of pigments like PA that contribute to the colors of fruits, and in defending against pathogens and environmental stresses. They are crucial for fruit development, ripening, structural maintenance in plants, and adaptation to various environmental factors. As such, these genes and enzymes are essential for plant growth and development, as well as for various biotechnological applications in environmental remediation and industrial processes. This review article emphasizes the significance of genes encoding laccase enzymes during fruit growth, specifically pertaining to the strengthening of the endocarp through lignification. This process is crucial for ensuring fruit defense and optimizing seed scattering. The information gathered in this article will aid breeders in producing future fruit-bearing plants that are resistant to disease, cost-effective, and nutrient-rich.
Asunto(s)
Frutas , Lignina , Lignina/metabolismo , Lacasa/metabolismo , Operón Lac , Semillas/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
The lactose uptake pathway of E. coli is a paradigmatic example of multistability in gene regulatory circuits. In the induced state of the lac pathway, the genes comprising the lac operon are transcribed, leading to the production of proteins that import and metabolize lactose. In the uninduced state, a stable repressor-DNA loop frequently blocks the transcription of the lac genes. Transitions from one phenotypic state to the other are driven by fluctuations, which arise from the random timing of the binding of ligands and proteins. This stochasticity affects transcription and translation, and ultimately molecular copy numbers. Our aim is to understand the transition from the induced to the uninduced state of the lac operon. We use a detailed computational model to show that repressor-operator binding and unbinding, fluctuations in the total number of repressors, and inducer-repressor binding and unbinding all play a role in this transition. Based on the timescales on which these processes operate, we construct a minimal model of the transition to the uninduced state and compare the results with simulations and experimental observations. The induced state turns out to be very stable, with a transition rate to the uninduced state lower than 2×10-9 per minute. In contrast to the transition to the induced state, the transition to the uninduced state is well described in terms of a 2D diffusive system crossing a barrier, with the diffusion rates emerging from a model of repressor unbinding.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Operón Lac , Represoras Lac/genética , Represoras Lac/metabolismo , Lactosa/metabolismoRESUMEN
The lac operon of Escherichia coli is repressed several 100-fold in the presence of glucose. This repression has been attributed to cAMP receptor protein-mediated inhibition of lac transcription and EIIAGlc-mediated inhibition of lactose transport (inducer exclusion). The growing evidence against the first mechanism has led to the postulate that the repression is driven by inducer exclusion. Although inducer exclusion reduces the permease activity only 2-fold in fully induced cells, it could be more potent in partially induced cells. Here, we show that even in partially induced cells, inducer exclusion reduces the permease activity no more than 6-fold. Moreover, the repression is so small because these experiments are performed in the presence of chloramphenicol. Indeed, when glucose is added to a culture growing on glycerol and TMG, but no chloramphenicol, lac expression is repressed 900-fold. This repression is primarily due to reversal of the positive feedback loop, i.e., the decline of the intracellular TMG level leads to a lower permease level, which reduces the intracellular TMG level even further. The repression in the absence of chloramphenicol is therefore primarily due to positive feedback, which does not exist during measurements of inducer exclusion.