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1.
Mol Cell ; 81(17): 3560-3575.e6, 2021 09 02.
Artículo en Inglés | MEDLINE | ID: mdl-34375585

RESUMEN

Transcription initiation by RNA polymerase II (RNA Pol II) requires preinitiation complex (PIC) assembly at gene promoters. In the dynamic nucleus, where thousands of promoters are broadly distributed in chromatin, it is unclear how multiple individual components converge on any target to establish the PIC. Here we use live-cell, single-molecule tracking in S. cerevisiae to visualize constrained exploration of the nucleoplasm by PIC components and Mediator's key role in guiding this process. On chromatin, TFIID/TATA-binding protein (TBP), Mediator, and RNA Pol II instruct assembly of a short-lived PIC, which occurs infrequently but efficiently within a few seconds on average. Moreover, PIC exclusion by nucleosome encroachment underscores regulated promoter accessibility by chromatin remodeling. Thus, coordinated nuclear exploration and recruitment to accessible targets underlies dynamic PIC establishment in yeast. Our study provides a global spatiotemporal model for transcription initiation in live cells.


Asunto(s)
Complejo Mediador/metabolismo , ARN Polimerasa II/metabolismo , Iniciación de la Transcripción Genética/fisiología , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/fisiología , Complejo Mediador/genética , Nucleosomas/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Análisis Espacio-Temporal , Proteína de Unión a TATA-Box/genética , Factor de Transcripción TFIID/genética , Transcripción Genética/genética
2.
Elife ; 92020 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-32338606

RESUMEN

The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription at yeast promoters is responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.


Asunto(s)
Histonas/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Transcripción Genética , Ensamble y Desensamble de Cromatina , Histonas/genética , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , Proteínas de Saccharomyces cerevisiae/genética , Imagen Individual de Molécula , Sitio de Iniciación de la Transcripción
3.
Elife ; 4: e06845, 2015 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-26116819

RESUMEN

The histone variant H2A.Z is a universal mark of gene promoters, enhancers, and regulatory elements in eukaryotic chromatin. The chromatin remodeler SWR1 mediates site-specific incorporation of H2A.Z by a multi-step histone replacement reaction, evicting histone H2A-H2B from the canonical nucleosome and depositing the H2A.Z-H2B dimer. Binding of both substrates, the canonical nucleosome and the H2A.Z-H2B dimer, is essential for activation of SWR1. We found that SWR1 primarily recognizes key residues within the α2 helix in the histone-fold of nucleosomal histone H2A, a region not previously known to influence remodeler activity. Moreover, SWR1 interacts preferentially with nucleosomal DNA at superhelix location 2 on the nucleosome face distal to its linker-binding site. Our findings provide new molecular insights on recognition of the canonical nucleosome by a chromatin remodeler and have implications for ATP-driven mechanisms of histone eviction and deposition.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Ensamble y Desensamble de Cromatina , ADN/metabolismo , Histonas/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromatina/metabolismo , Unión Proteica , Saccharomyces cerevisiae/genética
4.
Elife ; 3: e02203, 2014 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-24844245

RESUMEN

The budding yeast centromere contains Cse4, a specialized histone H3 variant. Fluorescence pulse-chase analysis of an internally tagged Cse4 reveals that it is replaced with newly synthesized molecules in S phase, remaining stably associated with centromeres thereafter. In contrast, C-terminally-tagged Cse4 is functionally impaired, showing slow cell growth, cell lethality at elevated temperatures, and extra-centromeric nuclear accumulation. Recent studies using such strains gave conflicting findings regarding the centromeric abundance and cell cycle dynamics of Cse4. Our findings indicate that internally tagged Cse4 is a better reporter of the biology of this histone variant. Furthermore, the size of centromeric Cse4 clusters was precisely mapped with a new 3D-PALM method, revealing substantial compaction during anaphase. Cse4-specific chaperone Scm3 displays steady-state, stoichiometric co-localization with Cse4 at centromeres throughout the cell cycle, while undergoing exchange with a nuclear pool. These findings suggest that a stable Cse4 nucleosome is maintained by dynamic chaperone-in-residence Scm3.DOI: http://dx.doi.org/10.7554/eLife.02203.001.


Asunto(s)
Centrómero , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Fase S , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Anafase , Replicación del ADN , ADN de Hongos/biosíntesis , Fluorescencia , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética
5.
Cell ; 154(6): 1232-45, 2013 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-24034247

RESUMEN

The histone variant H2A.Z is a genome-wide signature of nucleosomes proximal to eukaryotic regulatory DNA. Whereas the multisubunit chromatin remodeler SWR1 is known to catalyze ATP-dependent deposition of H2A.Z, the mechanism of SWR1 recruitment to S. cerevisiae promoters has been unclear. A sensitive assay for competitive binding of dinucleosome substrates revealed that SWR1 preferentially binds long nucleosome-free DNA and the adjoining nucleosome core particle, allowing discrimination of gene promoters over gene bodies. Analysis of mutants indicates that the conserved Swc2/YL1 subunit and the adenosine triphosphatase domain of Swr1 are mainly responsible for binding to substrate. SWR1 binding is enhanced on nucleosomes acetylated by the NuA4 histone acetyltransferase, but recognition of nucleosome-free and nucleosomal DNA is dominant over interaction with acetylated histones. Such hierarchical cooperation between DNA and histone signals expands the dynamic range of genetic switches, unifying classical gene regulation by DNA-binding factors with ATP-dependent nucleosome remodeling and posttranslational histone modifications.


Asunto(s)
Ensamble y Desensamble de Cromatina , Histonas/metabolismo , Complejos Multiproteicos/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilación , Adenosina Trifosfatasas/metabolismo , Secuencia de Bases , Histona Acetiltransferasas/metabolismo , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/genética
6.
Nat Methods ; 10(1): 60-3, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23223154

RESUMEN

Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z scanning and is often too slow to capture biological events. We report an aberration-corrected multifocus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multifocus system enables high-resolution 3D imaging in multiple colors with single-molecule sensitivity, at speeds limited by the camera readout time of a single image.


Asunto(s)
Caenorhabditis elegans/citología , Rastreo Celular , Imagenología Tridimensional/métodos , Microscopía Fluorescente , Neuronas/citología , Saccharomyces cerevisiae/citología , Animales , Neoplasias Óseas/enzimología , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Osteosarcoma/enzimología , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
7.
Methods Enzymol ; 512: 275-91, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22910211

RESUMEN

The evolutionarily conserved histone variant H2A.Z has an important role in the regulation of gene expression and the establishment of a buffer to the spread of silent heterochromatin. Saccharomyces cerevisiae Swr1, a Swi2/Snf2-related ATPase, is the catalytic core of a multisubunit chromatin remodeling enzyme, called the SWR1 complex, that efficiently replaces conventional histone H2A in nucleosomes with histone H2A.Z. Swr1 is required for the deposition of histone H2A.Z at stereotypical promoter locations in vivo, and Swr1 and H2A.Z commonly regulate a subset of yeast genes. Here, we describe an integrated nucleosome assembly-histone replacement system whereby histone exchange by chromatin remodeling activities may be analyzed in vitro. The system demonstrates ATP- and SWR1-complex-dependent replacement of histone H2A for histone H2A.Z on a preassembled nucleosome array. This system may also be adapted to analyze dynamic interactions between chromatin remodeling and modifying enzymes, histone chaperones, and nucleosome substrates containing canonical, variant, or covalently modified histones.


Asunto(s)
Adenosina Trifosfatasas/química , Ensamble y Desensamble de Cromatina , Histonas/química , Nucleosomas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae , Adenosina Trifosfatasas/aislamiento & purificación , Centrifugación por Gradiente de Densidad , ADN/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/aislamiento & purificación , Histonas/aislamiento & purificación , Ácidos Nucleicos Inmovilizados/química , Proteína 1 de Ensamblaje de Nucleosomas/química , Proteína 1 de Ensamblaje de Nucleosomas/aislamiento & purificación , Plásmidos/química , Unión Proteica , Mapeo de Interacción de Proteínas , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación
8.
Mol Cell ; 43(3): 369-80, 2011 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-21816344

RESUMEN

The molecular architecture of centromere-specific nucleosomes containing histone variant CenH3 is controversial. We have biochemically reconstituted two distinct populations of nucleosomes containing Saccharomyces cerevisiae CenH3 (Cse4). Reconstitution of octameric nucleosomes containing histones Cse4/H4/H2A/H2B is robust on noncentromere DNA, but inefficient on AT-rich centromere DNA. However, nonhistone Scm3, which is required for Cse4 deposition in vivo, facilitates in vitro reconstitution of Cse4/H4/Scm3 complexes on AT-rich centromere sequences. Scm3 has a nonspecific DNA binding domain that shows preference for AT-rich DNA and a histone chaperone domain that promotes specific loading of Cse4/H4. In live cells, Scm3-GFP is enriched at centromeres in all cell cycle phases. Chromatin immunoprecipitation confirms that Scm3 occupies centromere DNA throughout the cell cycle, even when Cse4 and H4 are temporarily dislodged in S phase. These findings suggest a model in which centromere-bound Scm3 aids recruitment of Cse4/H4 to assemble and maintain an H2A/H2B-deficient centromeric nucleosome.


Asunto(s)
Centrómero/química , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/fisiología , Proteínas de Unión al ADN/química , Histonas/química , Nucleosomas/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/genética , Secuencia Rica en At , Sitios de Unión , Ciclo Celular/genética , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Fúngicos/metabolismo , ADN de Hongos/química , Proteínas de Unión al ADN/metabolismo , Chaperonas de Histonas/química , Chaperonas de Histonas/metabolismo , Chaperonas de Histonas/fisiología , Histonas/metabolismo , Modelos Moleculares , Nucleosomas/metabolismo , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
Cell ; 143(5): 725-36, 2010 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-21111233

RESUMEN

Histone variant H2A.Z-containing nucleosomes are incorporated at most eukaryotic promoters. This incorporation is mediated by the conserved SWR1 complex, which replaces histone H2A in canonical nucleosomes with H2A.Z in an ATP-dependent manner. Here, we show that promoter-proximal nucleosomes are highly heterogeneous for H2A.Z in Saccharomyces cerevisiae, with substantial representation of nucleosomes containing one, two, or zero H2A.Z molecules. SWR1-catalyzed H2A.Z replacement in vitro occurs in a stepwise and unidirectional fashion, one H2A.Z-H2B dimer at a time, producing heterotypic nucleosomes as intermediates and homotypic H2A.Z nucleosomes as end products. The ATPase activity of SWR1 is specifically stimulated by H2A-containing nucleosomes without ensuing histone H2A eviction. Remarkably, further addition of free H2A.Z-H2B dimer leads to hyperstimulation of ATPase activity, eviction of nucleosomal H2A-H2B, and deposition of H2A.Z-H2B. These results suggest that the combination of H2A-containing nucleosome and free H2A.Z-H2B dimer acting as both effector and substrate for SWR1 governs the specificity and outcome of the replacement reaction.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Ensamble y Desensamble de Cromatina , Histonas/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Dimerización , Histonas/química , Histonas/genética , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
10.
J Biol Chem ; 284(10): 6200-7, 2009 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-19088068

RESUMEN

Variant histone H2AZ-containing nucleosomes are involved in the regulation of gene expression. In Saccharomyces cerevisiae, chromatin deposition of histone H2AZ is mediated by the fourteen-subunit SWR1 complex, which catalyzes ATP-dependent exchange of nucleosomal histone H2A for H2AZ. Previous work defined the role of seven SWR1 subunits (Swr1 ATPase, Swc2, Swc3, Arp6, Swc5, Yaf9, and Swc6) in maintaining complex integrity and H2AZ histone replacement activity. Here we examined the function of three additional SWR1 subunits, bromodomain containing Bdf1, actin-related protein Arp4 and Swc7, by analyzing affinity-purified mutant SWR1 complexes. We observed that depletion of Arp4 (arp4-td) substantially impaired the association of Bdf1, Yaf9, and Swc4. In contrast, loss of either Bdf1 or Swc7 had minimal effects on overall complex integrity. Furthermore, the basic H2AZ histone replacement activity of SWR1 in vitro required Arp4, but not Bdf1 or Swc7. Thus, three out of fourteen SWR1 subunits, Bdf1, Swc7, and previously noted Swc3, appear to have roles auxiliary to the basic histone replacement activity. The N-terminal region of the Swr1 ATPase subunit is necessary and sufficient to direct association of Bdf1 and Swc7, as well as Arp4, Act1, Yaf9 and Swc4. This same region contains an additional H2AZ-H2B specific binding site, distinct from the previously identified Swc2 subunit. These findings suggest that one SWR1 enzyme might be capable of binding two H2AZ-H2B dimers, and provide further insight on the hierarchy and interdependency of molecular interactions within the SWR1 complex.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Ensamble y Desensamble de Cromatina/fisiología , Histonas/metabolismo , Complejos Multiproteicos/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Actinas/genética , Actinas/metabolismo , Adenosina Trifosfatasas/genética , Histona Acetiltransferasas , Histonas/genética , Complejos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleosomas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
11.
Cell ; 129(6): 1153-64, 2007 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-17574026

RESUMEN

The budding yeast histone H3 variant, Cse4, replaces conventional histone H3 in centromeric chromatin and, together with centromere-specific DNA-binding factors, directs assembly of the kinetochore, a multiprotein complex mediating chromosome segregation. We have identified Scm3, a nonhistone protein that colocalizes with Cse4 and is required for its centromeric association. Bacterially expressed Scm3 binds directly to and reconstitutes a stoichiometric complex with Cse4 and histone H4 but not with conventional histone H3 and H4. A conserved acidic domain of Scm3 is responsible for directing the Cse4-specific interaction. Strikingly, binding of Scm3 can replace histones H2A-H2B from preassembled Cse4-containing histone octamers. This incompatibility between Scm3 and histones H2A-H2B is correlated with diminished in vivo occupancy of histone H2B, H2A, and H2AZ at centromeres. Our findings indicate that nonhistone Scm3 serves to assemble and maintain Cse4-H4 at centromeres and may replace histone H2A-H2B dimers in a centromere-specific nucleosome core.


Asunto(s)
Centrómero/química , Proteínas Cromosómicas no Histona/fisiología , Proteínas de Unión al ADN/fisiología , Histonas/metabolismo , Nucleosomas/química , Proteínas de Saccharomyces cerevisiae/fisiología , Ciclo Celular , Dimerización , Histonas/química , Cinetocoros/química , Cinetocoros/metabolismo , Modelos Biológicos , Modelos Genéticos , Unión Proteica , Proteínas Recombinantes/química , Temperatura
12.
Mol Cell ; 25(3): 357-68, 2007 Feb 09.
Artículo en Inglés | MEDLINE | ID: mdl-17289584

RESUMEN

The histone variant H2AZ marks nucleosomes flanking the promoters of most genes of budding yeast. The incorporation of H2AZ into chromatin is dependent on the SWR1 complex, which catalyses the replacement of conventional histone H2A with H2AZ. In cells, the pool of unincorporated histone H2AZ has previously been found in association with Nap1, a chaperone for conventional histone H2A-H2B. Here, we report the discovery of Chz1, a histone chaperone that has preference for H2AZ and can also deliver a source of the histone variant for SWR1-dependent histone replacement. Bacterially expressed Chz1 forms a heterotrimer with H2AZ-H2B, stabilizing the association of the histone dimer. We have identified a conserved motif important for histone variant recognition within the H2AZ-interacting domain of Chz1. The presence of this motif in other metazoan proteins suggests that H2AZ-specific chaperones may be widely conserved.


Asunto(s)
Histonas/metabolismo , Chaperonas Moleculares/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Proteínas Portadoras/química , Proteínas de Ciclo Celular/fisiología , Secuencia Conservada , Dimerización , Chaperonas de Histonas , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Proteínas Nucleares/fisiología , Proteína 1 de Ensamblaje de Nucleosomas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia
13.
Nat Struct Mol Biol ; 12(12): 1064-71, 2005 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-16299513

RESUMEN

The histone variant H2AZ is incorporated preferentially at specific locations in chromatin to modulate chromosome functions. In Saccharomyces cerevisiae, deposition of histone H2AZ is mediated by the multiprotein SWR1 complex, which catalyzes ATP-dependent exchange of nucleosomal histone H2A for H2AZ. Here, we define interactions between SWR1 components and H2AZ, revealing a link between the ATPase domain of Swr1 and three subunits required for the binding of H2AZ. We discovered that Swc2 binds directly to and is essential for transfer of H2AZ. Swc6 and Arp6 are necessary for the association of Swc2 and for nucleosome binding, whereas other subunits, Swc5 and Yaf9, are required for H2AZ transfer but neither H2AZ nor nucleosome binding. Finally, the C-terminal alpha-helix of H2AZ is crucial for its recognition by SWR1. These findings provide insight on the initial events of histone exchange.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Secuencia Conservada , Histonas/química , Histonas/genética , Datos de Secuencia Molecular , Mutación , Nucleosomas/metabolismo , Estructura Secundaria de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
14.
Science ; 303(5656): 343-8, 2004 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-14645854

RESUMEN

The conserved histone variant H2AZ has an important role in the regulation of gene expression and the establishment of a buffer to the spread of silent heterochromatin. How histone variants such as H2AZ are incorporated into nucleosomes has been obscure. We have found that Swr1, a Swi2/Snf2-related adenosine triphosphatase, is the catalytic core of a multisubunit, histone-variant exchanger that efficiently replaces conventional histone H2A with histone H2AZ in nucleosome arrays. Swr1 is required for the deposition of histone H2AZ at specific chromosome locations in vivo, and Swr1 and H2AZ commonly regulate a subset of yeast genes. These findings define a previously unknown role for the adenosine triphosphate-dependent chromatin remodeling machinery.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Cromatina/metabolismo , Regulación Fúngica de la Expresión Génica , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/aislamiento & purificación , Catálisis , Dominio Catalítico , Cromosomas Fúngicos/genética , ADN de Hongos/genética , ADN de Hongos/metabolismo , Dimerización , Perfilación de la Expresión Génica , Silenciador del Gen , Genes Fúngicos , Histonas/genética , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Telómero/genética , Transcripción Genética
15.
J Biol Chem ; 277(30): 27154-61, 2002 Jul 26.
Artículo en Inglés | MEDLINE | ID: mdl-12021283

RESUMEN

The Mediator complex is generally required for transcriptional regulation in species ranging from yeast to human. Throughout evolution, the functional diversity of the Mediator complex has been enhanced to meet the increasing requirements for sophisticated gene regulation. It is likely that greater structural complexity is thus required to accomplish these new, complex regulatory functions. In this study, we took systematic steps to examine various types of Mediator complexes in Drosophila melanogaster. Such efforts led to the identification of three distinct forms of Mediator complexes. In exploring their compositional and functional heterogeneity, we found that the smallest complex (C1) is highly enriched in a certain type of Drosophila cells and possesses novel Mediator proteins. The subunits shared among the three Mediator complexes (C1, C2, and C3) appear to form a stable modular structure that serves as a binding surface for transcriptional activator proteins. However, only C2 and C3 were able to support activated transcription in vitro. These findings suggest that different cell types may require distinct Mediator complexes, some of which may participate in nuclear processes other than the previously identified functions.


Asunto(s)
Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Animales , Western Blotting , Línea Celular , Núcleo Celular/metabolismo , Cromatografía en Gel , Medio de Cultivo Libre de Suero , Drosophila melanogaster , Electroforesis en Gel de Poliacrilamida , Luciferasas/metabolismo , Datos de Secuencia Molecular , Unión Proteica , ARN/metabolismo , ARN Bicatenario
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