RESUMEN
The electron transport chain (ETC) is a well-studied and highly conserved metabolic pathway that produces ATP through generation of a proton gradient across the inner mitochondrial membrane coupled to oxidative phosphorylation. ETC mutations are associated with a wide array of human disease conditions and to aging-related phenotypes in a number of different organisms. In this study, we sought to better understand the role of the ETC in aging using a yeast model. A panel of ETC mutant strains that fail to survive starvation was used to isolate suppressor mutants that survive. These suppressors tend to fall into major nutrient sensing and signaling pathways, suggesting that the ETC is involved in proper starvation signaling to these pathways in yeast. These suppressors also partially restore ETC-associated gene expression and pH homeostasis defects, though it remains unclear whether these phenotypes directly cause the suppression or are simply effects. This work further highlights the complex cellular network connections between metabolic pathways and signaling events in the cell and their potential roles in aging and age-related diseases.
Asunto(s)
Transporte de Electrón/genética , Mutación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Citosol/química , Citosol/metabolismo , Transporte de Electrón/fisiología , Regulación Fúngica de la Expresión Génica , Genoma Mitocondrial , Glucosa/metabolismo , Concentración de Iones de Hidrógeno , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Factores de Transcripción/genéticaRESUMEN
The ability to switch a gene from off to on and monitor dynamic changes provides a powerful approach for probing gene function and elucidating causal regulatory relationships. Here, we developed and characterized YETI (Yeast Estradiol strains with Titratable Induction), a collection in which > 5,600 yeast genes are engineered for transcriptional inducibility with single-gene precision at their native loci and without plasmids. Each strain contains SGA screening markers and a unique barcode, enabling high-throughput genetics. We characterized YETI using growth phenotyping and BAR-seq screens, and we used a YETI allele to identify the regulon of Rof1, showing that it acts to repress transcription. We observed that strains with inducible essential genes that have low native expression can often grow without inducer. Analysis of data from eukaryotic and prokaryotic systems shows that native expression is a variable that can bias promoter-perturbing screens, including CRISPRi. We engineered a second expression system, Z3 EB42, that gives lower expression than Z3 EV, a feature enabling conditional activation and repression of lowly expressed essential genes that grow without inducer in the YETI library.
Asunto(s)
Genes Esenciales , Saccharomyces cerevisiae , Biblioteca de Genes , Plásmidos , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genéticaRESUMEN
Trehalose metabolism in yeast has been linked to a variety of phenotypes, including heat resistance, desiccation tolerance, carbon-source utilization, and sporulation. The relationships among the several phenotypes of mutants unable to synthesize trehalose are not understood, even though the pathway is highly conserved. One of these phenotypes is that tps1Δ strains cannot reportedly grow on media containing glucose or fructose, even when another carbon source they can use (e.g. galactose) is present. Here we corroborate the recent observation that a small fraction of yeast tps1Δ cells do grow on glucose, unlike the majority of the population. This is not due to a genetic alteration, but instead resembles the persister phenotype documented in many microorganisms and cancer cells undergoing lethal stress. We extend these observations to show that this phenomenon is glucose-specific, as it does not occur on another highly fermented carbon source, fructose. We further demonstrate that this phenomenon appears to be related to mitochondrial complex III function, but unrelated to inorganic phosphate levels in the cell, as had previously been suggested. Finally, we found that this phenomenon is specific to S288C-derived strains, and is the consequence of a variant in the MKT1 gene.
Asunto(s)
Glucosa/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Complejo III de Transporte de Electrones/metabolismo , Fermentación , Fructosa/metabolismo , Glucosiltransferasas/genética , Mutación con Pérdida de Función , Trehalosa/biosíntesisRESUMEN
We present IDEA (the Induction Dynamics gene Expression Atlas), a dataset constructed by independently inducing hundreds of transcription factors (TFs) and measuring timecourses of the resulting gene expression responses in budding yeast. Each experiment captures a regulatory cascade connecting a single induced regulator to the genes it causally regulates. We discuss the regulatory cascade of a single TF, Aft1, in detail; however, IDEA contains > 200 TF induction experiments with 20 million individual observations and 100,000 signal-containing dynamic responses. As an application of IDEA, we integrate all timecourses into a whole-cell transcriptional model, which is used to predict and validate multiple new and underappreciated transcriptional regulators. We also find that the magnitudes of coefficients in this model are predictive of genetic interaction profile similarities. In addition to being a resource for exploring regulatory connectivity between TFs and their target genes, our modeling approach shows that combining rapid perturbations of individual genes with genome-scale time-series measurements is an effective strategy for elucidating gene regulatory networks.
Asunto(s)
Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Saccharomycetales/genética , Factores de Transcripción/genética , Algoritmos , Bases de Datos Genéticas , Proteínas Fúngicas/genética , Regulación de la Expresión GénicaRESUMEN
A high-confidence map of the direct, functional targets of each transcription factor (TF) requires convergent evidence from independent sources. Two significant sources of evidence are TF binding locations and the transcriptional responses to direct TF perturbations. Systematic data sets of both types exist for yeast and human, but they rarely converge on a common set of direct, functional targets for a TF. Even the few genes that are both bound and responsive may not be direct functional targets. Our analysis shows that when there are many nonfunctional binding sites and many indirect targets, nonfunctional sites are expected to occur in the cis-regulatory DNA of indirect targets by chance. To address this problem, we introduce dual threshold optimization (DTO), a new method for setting significance thresholds on binding and perturbation-response data, and show that it improves convergence. It also enables comparison of binding data to perturbation-response data that have been processed by network inference algorithms, which further improves convergence. The combination of dual threshold optimization and network inference greatly expands the high-confidence TF network map in both yeast and human. Next, we analyze a comprehensive new data set measuring the transcriptional response shortly after inducing overexpression of a yeast TF. We also present a new yeast binding location data set obtained by transposon calling cards and compare it to recent ChIP-exo data. These new data sets improve convergence and expand the high-confidence network synergistically.
Asunto(s)
Factores de Transcripción/metabolismo , Algoritmos , Sitios de Unión , Secuenciación de Inmunoprecipitación de Cromatina , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Células HEK293 , Humanos , Células K562 , Factores de Transcripción/genética , Transcripción Genética , Levaduras/genética , Levaduras/metabolismoRESUMEN
Anthozoa-class red fluorescent proteins (RFPs) are frequently used as biological markers, with far-red (λem â¼ 600-700 nm) emitting variants sought for whole-animal imaging because biological tissues are more permeable to light in this range. A barrier to the use of naturally occurring RFP variants as molecular markers is that all are tetrameric, which is not ideal for cell biological applications. Efforts to engineer monomeric RFPs have typically produced dimmer and blue-shifted variants because the chromophore is sensitive to small structural perturbations. In fact, despite much effort, only four native RFPs have been successfully monomerized, leaving the majority of RFP biodiversity untapped in biomarker development. Here we report the generation of monomeric variants of HcRed and mCardinal, both far-red dimers, and describe a comprehensive methodology for the monomerization of red-shifted oligomeric RFPs. Among the resultant variants is mKelly1 (emission maximum, λem = 656 nm), which, along with the recently reported mGarnet2 [Matela G, et al. (2017) Chem Commun (Camb) 53:979-982], forms a class of bright, monomeric, far-red FPs.
Asunto(s)
Antozoos/metabolismo , Proteínas Luminiscentes/química , Animales , Antozoos/química , Antozoos/genética , Color , Cristalografía por Rayos X , Fluorescencia , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Modelos Moleculares , Ingeniería de Proteínas , Proteína Fluorescente RojaRESUMEN
Replicative aging of Saccharomyces cerevisiae is an established model system for eukaryotic cellular aging. A limitation in yeast lifespan studies has been the difficulty of separating old cells from young cells in large quantities. We engineered a new platform, the Miniature-chemostat Aging Device (MAD), that enables purification of aged cells at sufficient quantities for genomic and biochemical characterization of aging yeast populations. Using MAD, we measured DNA accessibility and gene expression changes in aging cells. Our data highlight an intimate connection between aging, growth rate, and stress. Stress-independent genes that change with age are highly enriched for targets of the signal recognition particle (SRP). Combining MAD with an improved ATAC-seq method, we find that increasing proteasome activity reduces rDNA instability usually observed in aging cells and, contrary to published findings, provide evidence that global nucleosome occupancy does not change significantly with age.
Asunto(s)
Cromatina/metabolismo , Replicación del ADN , Técnicas Microbiológicas/métodos , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/aislamiento & purificación , Perfilación de la Expresión Génica , Análisis de Secuencia de ARNRESUMEN
Animals have evolved to survive, and even thrive, in different environments. Genetic adaptations may have indirectly created phenotypes that also resulted in a longer lifespan. One example of this phenomenon is the preternaturally long-lived naked mole-rat. This strictly subterranean rodent tolerates hypoxia, hypercapnia, and soil-based toxins. Naked mole-rats also exhibit pronounced resistance to cancer and an attenuated decline of many physiological characteristics that often decline as mammals age. Elucidating mechanisms that give rise to their unique phenotypes will lead to better understanding of subterranean ecophysiology and biology of aging. Comparative genomics could be a useful tool in this regard. Since the publication of a naked mole-rat genome assembly in 2011, analyses of genomic and transcriptomic data have enabled a clearer understanding of mole-rat evolutionary history and suggested molecular pathways (e.g., NRF2-signaling activation and DNA damage repair mechanisms) that may explain the extraordinarily longevity and unique health traits of this species. However, careful scrutiny and re-analysis suggest that some identified features result from incorrect or imprecise annotation and assembly of the naked mole-rat genome: in addition, some of these conclusions (e.g., genes involved in cancer resistance and hairlessness) are rejected when the analysis includes additional, more closely related species. We describe how the combination of better study design, improved genomic sequencing techniques, and new bioinformatic and data analytical tools will improve comparative genomics and ultimately bridge the gap between traditional model and nonmodel organisms.
Asunto(s)
Envejecimiento/genética , Genoma , Genómica , Longevidad/genética , Animales , Mamíferos/genética , Ratas Topo , Anotación de Secuencia Molecular , Ratas , Especificidad de la Especie , Transcriptoma/genéticaRESUMEN
Methionine restriction is a widely reported intervention for increasing life span in several model organisms. Low circulating levels of methionine are evident in the long-lived naked mole-rat, suggesting that it naturally presents with a life-extending phenotype akin to that observed in methionine-restricted animals. Similarly, long-lived dwarf mice also appear to have altered methionine metabolism. The mechanisms underlying methionine-restriction effects on life-span extension, however, remain unknown, as do their potential connections with caloric restriction, another well-established intervention for prolonging life span. Paradoxically, methionine is enriched in proteins expressed in mitochondria and may itself serve an important role in the detoxification of reactive oxygen species and may thereby contribute to delayed aging. Collectively, we highlight the evidence that modulation of the methionine metabolic network can extend life span-from yeast to humans-and explore the evidence that sulfur amino acids and the concomitant transsulfuration pathway play a privileged role in this regard. However, systematic studies in single organisms (particularly those that exhibit extreme longevity) are still required to distinguish the fundamental principles concerning the role of methionine and other amino acids in regulating life span.
Asunto(s)
Restricción Calórica , Esperanza de Vida , Longevidad , Metionina/metabolismo , Levaduras , Envejecimiento , Animales , Cisteína/metabolismo , Células Eucariotas/fisiología , Humanos , Invertebrados , Redes y Vías Metabólicas , Modelos Animales , Roedores , Levaduras/fisiologíaRESUMEN
Vinculin is filamentous (F)-actin-binding protein enriched in integrin-based adhesions to the extracellular matrix (ECM). Whereas studies in 2-dimensional (2D) tissue culture models have suggested that vinculin negatively regulates cell migration by promoting cytoskeleton-ECM coupling to strengthen and stabilize adhesions, its role in regulating cell migration in more physiologic, 3-dimensional (3D) environments is unclear. To address the role of vinculin in 3D cell migration, we analyzed the morphodynamics, migration, and ECM remodeling of primary murine embryonic fibroblasts (MEFs) with cre/loxP-mediated vinculin gene disruption in 3D collagen I cultures. We found that vinculin promoted 3D cell migration by increasing directional persistence. Vinculin was necessary for persistent cell protrusion, cell elongation, and stable cell orientation in 3D collagen, but was dispensable for lamellipodia formation, suggesting that vinculin-mediated cell adhesion to the ECM is needed to convert actin-based cell protrusion into persistent cell shape change and migration. Consistent with this finding, vinculin was necessary for efficient traction force generation in 3D collagen without affecting myosin II activity and promoted 3D collagen fiber alignment and macroscopical gel contraction. Our results suggest that vinculin promotes directionally persistent cell migration and tension-dependent ECM remodeling in complex 3D environments by increasing cell-ECM adhesion and traction force generation.
Asunto(s)
Movimiento Celular/fisiología , Polaridad Celular/fisiología , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Vinculina/metabolismo , Animales , Colágeno/genética , Matriz Extracelular/genética , Fibroblastos/citología , Ratones , Ratones Noqueados , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Seudópodos/genética , Seudópodos/metabolismo , Vinculina/genéticaRESUMEN
Protein engineering of microbial rhodopsins has been successful in generating variants with improved properties for applications in optogenetics. Members of this membrane protein family can act as both actuators and sensors of neuronal activity. Chimeragenesis, structure-guided mutagenesis, and directed evolution have proven effective strategies for tuning absorption wavelength, altering ion specificity and increasing fluorescence. These approaches facilitate the development of useful optogenetic tools and, in some cases, have yielded insights into rhodopsin structure-function relationships.
Asunto(s)
Optogenética/métodos , Ingeniería de Proteínas , Rodopsinas Microbianas/química , Animales , Técnicas Biosensibles , Electrofisiología , Mamíferos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Conformación ProteicaRESUMEN
Proton-pumping rhodopsins (PPRs) are photoactive retinal-binding proteins that transport ions across biological membranes in response to light. These proteins are interesting for light-harvesting applications in bioenergy production, in optogenetics applications in neuroscience, and as fluorescent sensors of membrane potential. Little is known, however, about how the protein sequence determines the considerable variation in spectral properties of PPRs from different biological niches or how to engineer these properties in a given PPR. Here we report a comprehensive study of amino acid substitutions in the retinal-binding pocket of Gloeobacter violaceus rhodopsin (GR) that tune its spectral properties. Directed evolution generated 70 GR variants with absorption maxima shifted by up to ±80nm, extending the protein's light absorption significantly beyond the range of known natural PPRs. While proton-pumping activity was disrupted in many of the spectrally shifted variants, we identified single tuning mutations that incurred blue and red shifts of 42nm and 22nm, respectively, that did not disrupt proton pumping. Blue-shifting mutations were distributed evenly along the retinal molecule while red-shifting mutations were clustered near the residue K257, which forms a covalent bond with retinal through a Schiff base linkage. Thirty eight of the identified tuning mutations are not found in known microbial rhodopsins. We discovered a subset of red-shifted GRs that exhibit high levels of fluorescence relative to the WT (wild-type) protein.
Asunto(s)
Cianobacterias/metabolismo , Evolución Molecular Dirigida , Bombas de Protones/metabolismo , Rodopsinas Microbianas/química , Rodopsinas Microbianas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Cianobacterias/genética , Cianobacterias/crecimiento & desarrollo , Fluorescencia , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación/genética , Rodopsinas Microbianas/genética , Homología de Secuencia de AminoácidoRESUMEN
Microbial rhodopsins are a diverse group of photoactive transmembrane proteins found in all three domains of life. A member of this protein family, Archaerhodopsin-3 (Arch) of halobacterium Halorubrum sodomense, was recently shown to function as a fluorescent indicator of membrane potential when expressed in mammalian neurons. Arch fluorescence, however, is very dim and is not optimal for applications in live-cell imaging. We used directed evolution to identify mutations that dramatically improve the absolute brightness of Arch, as confirmed biochemically and with live-cell imaging (in Escherichia coli and human embryonic kidney 293 cells). In some fluorescent Arch variants, the pK(a) of the protonated Schiff-base linkage to retinal is near neutral pH, a useful feature for voltage-sensing applications. These bright Arch variants enable labeling of biological membranes in the far-red/infrared and exhibit the furthest red-shifted fluorescence emission thus far reported for a fluorescent protein (maximal excitation/emission at â¼ 620 nm/730 nm).
Asunto(s)
Proteínas Arqueales/metabolismo , Evolución Molecular Dirigida , Sitios de Unión , Supervivencia Celular , Escherichia coli/metabolismo , Fluorescencia , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Halorubrum/metabolismo , Humanos , Proteínas Mutantes/metabolismo , Mutación , Homología Estructural de ProteínaRESUMEN
A conditional gene expression system that is fast-acting, is tunable and achieves single-gene specificity was recently developed for yeast. A gene placed directly downstream of a modified GAL1 promoter containing six Zif268 binding sequences (with single nucleotide spacing) was shown to be selectively inducible in the presence of ß-estradiol, so long as cells express the artificial transcription factor, Z3EV (a fusion of the Zif268 DNA binding domain, the ligand binding domain of the human estrogen receptor and viral protein 16). We show the strength of Z3EV-responsive promoters can be modified using straightforward design principles. By moving Zif268 binding sites toward the transcription start site, expression output can be nearly doubled. Despite the reported requirement of estrogen receptor dimerization for hormone-dependent activation, a single binding site suffices for target gene activation. Target gene expression levels correlate with promoter binding site copy number and we engineer a set of inducible promoter chassis with different input-output characteristics. Finally, the coupling between inducer identity and gene activation is flexible: the ligand specificity of Z3EV can be re-programmed to respond to a non-hormone small molecule with only five amino acid substitutions in the human estrogen receptor domain, which may prove useful for industrial applications.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Ingeniería Genética , Saccharomyces cerevisiae/genética , Regiones no Traducidas 5' , Sitios de Unión , Estradiol/farmacología , Galactoquinasa/genética , Ligandos , Regiones Promotoras Genéticas , Receptores de Estrógenos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Biología Sintética/métodos , Factores de Transcripción/metabolismoRESUMEN
Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.
Asunto(s)
Proteínas Fluorescentes Verdes/genética , Ingeniería de Proteínas/métodos , Biología Sintética/métodos , Factores de Transcripción/genética , Secuencia de Bases , Conexina 43/genética , Citometría de Flujo/métodos , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/química , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Plásmidos/genética , Factores de Transcripción/biosíntesis , Factores de Transcripción/química , Levaduras/genética , Levaduras/metabolismoRESUMEN
The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.
Asunto(s)
Hibridación Fluorescente in Situ/métodos , ARN Mensajero/análisis , Saccharomyces cerevisiae/genética , Algoritmos , ARN de Hongos/análisis , ARN de Hongos/química , ARN Mensajero/química , Saccharomyces cerevisiae/químicaRESUMEN
A general method for the dynamic control of single gene expression in eukaryotes, with no off-target effects, is a long-sought tool for molecular and systems biologists. We engineered two artificial transcription factors (ATFs) that contain Cys(2)His(2) zinc-finger DNA-binding domains of either the mouse transcription factor Zif268 (9 bp of specificity) or a rationally designed array of four zinc fingers (12 bp of specificity). These domains were expressed as fusions to the human estrogen receptor and VP16 activation domain. The ATFs can rapidly induce a single gene driven by a synthetic promoter in response to introduction of an otherwise inert hormone with no detectable off-target effects. In the absence of inducer, the synthetic promoter is inactive and the regulated gene product is not detected. Following addition of inducer, transcripts are induced >50-fold within 15 min. We present a quantitative characterization of these ATFs and provide constructs for making their implementation straightforward. These new tools allow for the elucidation of regulatory network elements dynamically, which we demonstrate with a major metabolic regulator, Gcn4p.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/química , Regulación de la Expresión Génica , Transcripción Genética , Dedos de Zinc , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Sitios de Unión , Proliferación Celular , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Estradiol/farmacología , Redes Reguladoras de Genes , Ingeniería Genética/métodos , Genoma Fúngico , Proteína Vmw65 de Virus del Herpes Simple/genética , Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Humanos , Ratones , Estructura Terciaria de Proteína , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Methionine abundance affects diverse cellular functions, including cell division, redox homeostasis, survival under starvation, and oxidative stress response. Regulation of the methionine biosynthetic pathway involves three DNA-binding proteins-Met31p, Met32p, and Cbf1p. We hypothesized that there exists a "division of labor" among these proteins that facilitates coordination of methionine biosynthesis with diverse biological processes. To explore combinatorial control in this regulatory circuit, we deleted CBF1, MET31, and MET32 individually and in combination in a strain lacking methionine synthase. We followed genome-wide gene expression as these strains were starved for methionine. Using a combination of bioinformatic methods, we found that these regulators control genes involved in biological processes downstream of sulfur assimilation; many of these processes had not previously been documented as methionine dependent. We also found that the different factors have overlapping but distinct functions. In particular, Met31p and Met32p are important in regulating methionine metabolism, whereas p functions as a "generalist" transcription factor that is not specific to methionine metabolism. In addition, Met31p and Met32p appear to regulate iron-sulfur cluster biogenesis through direct and indirect mechanisms and have distinguishable target specificities. Finally, CBF1 deletion sometimes has the opposite effect on gene expression from MET31 and MET32 deletion.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Proteínas de Unión al ADN , Metionina , Proteínas de Saccharomyces cerevisiae , Azufre/metabolismo , Factores de Transcripción , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/deficiencia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Metionina/biosíntesis , Metionina/genética , Metionina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Azufre/fisiología , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
In yeast, the pathways of sulfur assimilation are combinatorially controlled by five transcriptional regulators (three DNA-binding proteins [Met31p, Met32p, and Cbf1p], an activator [Met4p], and a cofactor [Met28p]) and a ubiquitin ligase subunit (Met30p). This regulatory system exerts combinatorial control not only over sulfur assimilation and methionine biosynthesis, but also on many other physiological functions in the cell. Recently we characterized a gene induction system that, upon the addition of an inducer, results in near-immediate transcription of a gene of interest under physiological conditions. We used this to perturb levels of single transcription factors during steady-state growth in chemostats, which facilitated distinction of direct from indirect effects of individual factors dynamically through quantification of the subsequent changes in genome-wide patterns of gene expression. We were able to show directly that Cbf1p acts sometimes as a repressor and sometimes as an activator. We also found circumstances in which Met31p/Met32p function as repressors, as well as those in which they function as activators. We elucidated and numerically modeled feedback relationships among the regulators, notably feedforward regulation of Met32p (but not Met31p) by Met4p that generates dynamic differences in abundance that can account for the differences in function of these two proteins despite their identical binding sites.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Metionina , Saccharomyces cerevisiae , Azufre/metabolismo , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/deficiencia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/fisiología , Sitios de Unión , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Genoma Fúngico , Metionina/biosíntesis , Metionina/genética , Metionina/metabolismo , Unión Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Azufre/fisiología , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Transcripción Genética , Ubiquitina/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismoRESUMEN
We describe the development and characterization of a system that allows the rapid and specific induction of individual genes in the yeast Saccharomyces cerevisiae without changes in nutrients or temperature. The system is based on the chimeric transcriptional activator Gal4dbd.ER.VP16 (GEV). Upon addition of the hormone ß-estradiol, cytoplasmic GEV localizes to the nucleus and binds to promoters containing Gal4p consensus binding sequences to activate transcription. With galactokinase Gal1p and transcriptional activator Gal4p absent, the system is fast-acting, resulting in readily detectable transcription within 5 min after addition of the inducer. ß-Estradiol is nearly a gratuitous inducer, as indicated by genome-wide profiling that shows unintended induction (by GEV) of only a few dozen genes. Response to inducer is graded: intermediate concentrations of inducer result in production of intermediate levels of product protein in all cells. We present data illustrating several applications of this system, including a modification of the regulated degron method, which allows rapid and specific degradation of a specific protein upon addition of ß-estradiol. These gene induction and protein degradation systems provide important tools for studying the dynamics and functional relationships of genes and their respective regulatory networks.