RESUMEN
The ParB-parS partition complexes that bacterial replicons use to ensure their faithful inheritance also find employment in visualization of DNA loci, as less intrusive alternatives to fluorescent repressor-operator systems. The ability of ParB molecules to interact via their N-terminal domains and to bind to non-specific DNA enables expansion of the initial complex to a size both functional in partition and, via fusion to fluorescent peptides, visible by light microscopy. We have investigated whether it is possible to dispense with the need to insert parS in the genomic locus of interest, by determining whether ParB fused to proteins that bind specifically to natural DNA sequences can still assemble visible complexes. In yeast cells, coproduction of fusions of ParB to a fluorescent peptide and to a TALE protein targeting an endogenous sequence did not yield visible foci; nor did any of several variants of these components. In E.coli, coproduction of fusions of SopB (F plasmid ParB) to fluorescent peptide, and to dCas9 together with specific guide RNAs, likewise yielded no foci. The result of coproducing analogous fusions of SopB proteins with distinct binding specificities was also negative. Our observations imply that in order to assemble higher order partition complexes, ParB proteins need specific activation through binding to their cognate parS sites.
Asunto(s)
Proteínas Bacterianas/metabolismo , Centrómero/química , Centrómero/metabolismo , ADN Bacteriano/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Bases , Sitios de Unión , Proteína 9 Asociada a CRISPR , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Plásmidos/genética , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Simportadores/genética , Simportadores/metabolismoRESUMEN
Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I.
Asunto(s)
Proteínas del Complejo de Iniciación de Transcripción Pol1/genética , ARN Polimerasa I/genética , ADN Ribosómico/genética , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , Precursores del ARN/genética , ARN Ribosómico , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Spatio-temporal organization of the cell nucleus adapts to and regulates genomic processes. Microscopy approaches that enable direct monitoring of specific chromatin sites in single cells and in real time are needed to better understand the dynamics involved. In this chapter, we describe the principle and development of ANCHOR, a novel tool for DNA labelling in eukaryotic cells. Protocols for use of ANCHOR to visualize a single genomic locus in eukaryotic cells are presented. We describe an approach for live cell imaging of a DNA locus during the entire cell cycle in human breast cancer cells.
Asunto(s)
ADN/química , Sitios Genéticos/genética , Microscopía Intravital/métodos , Imagen Molecular/métodos , Coloración y Etiquetado/métodos , Ciclo Celular/genética , Humanos , Imagenología Tridimensional/instrumentación , Imagenología Tridimensional/métodos , Microscopía Intravital/instrumentación , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Células MCF-7 , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Imagen Molecular/instrumentación , Transfección/instrumentación , Transfección/métodos , Transgenes/genéticaRESUMEN
Mating-type switching in yeast occurs through gene conversion between the MAT locus and one of two silent loci (HML or HMR) on opposite ends of the chromosome. MATa cells choose HML as template, whereas MATα cells use HMR. The recombination enhancer (RE) located on the left arm regulates this process. One long-standing hypothesis is that switching is guided by mating-type-specific and possibly RE-dependent chromosome folding. Here, we use Hi-C, 5C, and live-cell imaging to characterize the conformation of chromosome III in both mating types. We discovered a mating-type-specific conformational difference in the left arm. Deletion of a 1-kb subregion within the RE, which is not necessary during switching, abolished mating-type-dependent chromosome folding. The RE is therefore a composite element with one subregion essential for donor selection during switching and a separate region involved in modulating chromosome conformation.
Asunto(s)
Cromosomas Fúngicos/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Saccharomyces cerevisiae/genética , Cromatina/metabolismo , Cromosomas Fúngicos/química , Sitios Genéticos , Saccharomyces cerevisiae/metabolismoRESUMEN
It was recently observed that a glucose-enriched diet activates the insulin-like pathway in Caenorhabditis elegans, resulting in an inhibition of the FOXO transcription factor DAF-16. Because this signalling pathway is highly conserved from invertebrates to mammals and DAF-16 is a key player in innate immunity, we wondered whether a high-glucose diet, resembling the hyperglycaemic conditions in diabetic patients, would affect the susceptibility of C. elegans to bacterial pathogens isolated from different clinical situations (urinary tract or diabetic foot infections). We confirmed previous reports showing that such a diet decreases the lifespan of C. elegans fed with an avirulent Escherichia coli strain. However, glucose-fed nematodes appeared to be more resistant to most clinical isolates tested, showing that this invertebrate model does not mimic infections encountered in human diabetes, where patients show increased susceptibility to bacterial infections. This study also suggests that modulation of innate immunity in C. elegans, upon activation of the IGF1/insulin-like pathway by glucose, is not exclusively mediated by DAF-16, but also involves an additional factor that requires DAF-16 activity.