RESUMEN

With the identification of eight new polypeptides, we here complete the subunit characterization of the Schizosaccharomyces pombe RNA polymerase II holoenzyme. The complex contains homologs to all 10 essential gene products present in the Saccharomyces cerevisiae Mediator, but lacks clear homologs to any of the 10 S. cerevisiae components encoded by nonessential genes. S. pombe Mediator instead contains three unique components (Pmc2, -3, and -6), which lack homologs in other cell types. Presently, pmc2(+) and pmc3(+) have been shown to be nonessential genes. The data suggest that S. pombe and S. cerevisiae share an essential protein module, which associates with nonessential speciesspecific subunits. In support of this view, sequence analysis of the conserved yeast Mediator components Med4 and Med8 reveals sequence homology to the metazoan Mediator components Trap36 and Arc32. Therefore, 8 of 10 essential genes conserved between S. pombe and S. cerevisiae also have a metazoan homolog, indicating that an evolutionary conserved Mediator core is present in all eukaryotic cells. Our data suggest a closer functional relationship between yeast and metazoan Mediator than previously anticipated.

Asunto(s)

Secuencia Conservada , Proteínas Fúngicas/análisis , ARN Polimerasa II/análisis , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Transactivadores/análisis , Secuencia de Aminoácidos , Animales , Proteínas Fúngicas/genética , Holoenzimas/análisis , Humanos , Complejo Mediador , Datos de Secuencia Molecular , Proteínas Nucleares/análisis , Schizosaccharomyces/química , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrollo , Homología de Secuencia de Aminoácido , Transactivadores/genética , Factores de Transcripción/análisis , Activación Transcripcional , Levaduras

RESUMEN

The Mediator complex is essential for basal and regulated expression of nearly all RNA polymerase II-dependent genes in the Saccharomyces cerevisiae genome. Mediator acts as a bridge, conveying regulatory information from enhancers and other control elements to the promoter. It is now clear that Mediator-like complexes also exist in higher eukaryotic cells and that they have an important role in metazoan transcriptional regulation. However, the exact mechanism of Mediator-dependent transcriptional regulation remains unclear. We review here some recent advances in our understanding of Mediator structure and function. We also discuss a model to account for the functional and evolutionary relationship between yeast and metazoan Mediators. As an appendix to this review, we have created a database, MEDB, in which we have compiled information about all the S. cerevisiae Mediator subunits and their homologues in other eukaryotic cells (http://bio.lundberg.gu.se/medb/).

Asunto(s)

Proteínas Fúngicas , Regulación Fúngica de la Expresión Génica , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción , Activación Transcripcional , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

We have attempted to determine whether loss of mtDNA and respiratory chain function result in apoptosis in vivo. Apoptosis was studied in embryos with homozygous disruption of the mitochondrial transcription factor A gene (Tfam) and tissue-specific Tfam knockout animals with severe respiratory chain deficiency in the heart. We found massive apoptosis in Tfam knockout embryos at embryonic day (E) 9.5 and increased apoptosis in the heart of the tissue-specific Tfam knockouts. Furthermore, mtDNA-less (rho(0)) cell lines were susceptible to apoptosis induced by different stimuli in vitro. The data presented here provide in vivo evidence that respiratory chain deficiency predisposes cells to apoptosis, contrary to previous assumptions based on in vitro studies of cultured cells. These results suggest that increased apoptosis is a pathogenic event in human mtDNA mutation disorders. The finding that respiratory chain deficiency is associated with increased in vivo apoptosis may have important therapeutic implications for human disease. Respiratory chain deficiency and cell loss and/or apoptosis have been associated with neurodegeneration, heart failure, diabetes mellitus, and aging. Furthermore, chemotherapy and radiation treatment of cancer are intended to induce apoptosis in tumor cells. It would therefore be of interest to determine whether manipulation of respiratory chain function can be used to inhibit or enhance apoptosis in these conditions.

Asunto(s)

Apoptosis/genética , ADN Mitocondrial/genética , Proteínas de Unión al ADN , Expresión Génica , Proteínas del Grupo de Alta Movilidad , Proteínas Mitocondriales , Miocardio/metabolismo , Proteínas Nucleares , Transactivadores , Factores de Transcripción/metabolismo , Proteínas de Xenopus , Animales , Apoptosis/fisiología , Glutatión Peroxidasa/metabolismo , Humanos , Ratones , Ratones Noqueados , Miocardio/citología , Miocardio/enzimología , Oxidación-Reducción , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiología , Superóxido Dismutasa/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Células Tumorales Cultivadas , Regulación hacia Arriba

RESUMEN

Structures of yeast Mediator complex, of a related complex from mouse cells and of thyroid hormone receptor-associated protein complex from human cells have been determined by three-dimensional reconstruction from electron micrographs of single particles. All three complexes show a division in two parts, a "head" domain and a combined "middle-tail" domain. The head domains of the three complexes appear most similar and interact most closely with RNA polymerase II. The middle-tail domains show the greatest structural divergence and, in the case of the tail domain, may not interact with polymerase at all. Consistent with this structural divergence, analysis of a yeast Mediator mutant localizes subunits that are not conserved between yeast and mammalian cells to the tail domain. Biochemically defined Rgr1 and Srb4 modules of yeast Mediator are then assigned to the middle and head domains.

Asunto(s)

Proteínas Nucleares/ultraestructura , Receptores de Hormona Tiroidea/metabolismo , Transactivadores , Animales , Células HeLa , Humanos , Complejo Mediador , Ratones , Proteínas Nucleares/química , Conformación Proteica , Saccharomyces cerevisiae/química , Factores de Transcripción

RESUMEN

Mediator, a multiprotein complex involved in the regulation of RNA polymerase II transcription, binds to nucleosomes and acetylates histones. Three lines of evidence identify the Nut1 subunit of Mediator as responsible for the histone acetyltransferase (HAT) activity. An "in-gel" HAT assay reveals a single band of the appropriate size. Sequence alignment shows significant similarity of Nut1 to the GCN5-related N-acetyltransferase superfamily. Finally, recombinant Nut1 exhibits HAT activity in an in-gel assay.

Asunto(s)

Proteínas Fúngicas/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae , Acetiltransferasas/química , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Histona Acetiltransferasas , Sustancias Macromoleculares , Complejo Mediador , Datos de Secuencia Molecular , Complejos Multiproteicos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido

RESUMEN

A yeast strain harboring a temperature-sensitive allele of TFB3 (tfb3(ts)), the 38-kDa subunit of the RNA polymerase II transcription/nucleotide excision repair factor TFIIH, was found to be sensitive to ultraviolet (UV) radiation and defective for nucleotide excision repair in vitro. Interestingly, tfb3(ts) failed to grow on medium containing caffeine. A comprehensive pairwise two-hybrid analysis between yeast TFIIH subunits identified novel interactions between Rad3 and Tfb3, Tfb4 and Ssl1, as well as Ssl2 and Tfb2. These interactions have facilitated a more complete model of the structure of TFIIH and the nucleotide excision repairosome.

Asunto(s)

ADN Helicasas/metabolismo , Reparación del ADN , Proteínas de Saccharomyces cerevisiae , Factores Asociados con la Proteína de Unión a TATA , Factor de Transcripción TFIID , Factores de Transcripción TFII , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/metabolismo , Cafeína/farmacología , ADN Helicasas/química , Proteínas Fúngicas/metabolismo , Prueba de Complementación Genética , Modelos Biológicos , Mutación , Inhibidores de Fosfodiesterasa/farmacología , Plásmidos/metabolismo , Temperatura , Factor de Transcripción TFIIH , Factores de Transcripción/química , Técnicas del Sistema de Dos Híbridos , Rayos Ultravioleta

RESUMEN

We have purified the RNA polymerase II holoenzyme from Schizosaccharomyces pombe to near homogeneity. The Mediator complex is considerably smaller than its counterpart in Saccharomyces cerevisiae, containing only nine polypeptides larger than 19 kDa. Five of these Mediator subunits have been identified as the S. pombe homologs to Rgr1, Srb4, Med7, and Nut2 found in S. cerevisiae and the gene product of a previously uncharacterized open reading frame, PMC2, with no clear homologies to any described protein. The presence of Mediator in a S. pombe RNA polymerase II holoenzyme stimulated phosphorylation of the C-terminal domain by TFIIH purified from S. pombe. This stimulation was species-specific, because S. pombe Mediator could not stimulate TFIIH purified from S. cerevisiae. We suggest that the overall structure and mechanism of the Mediator is evolutionary conserved. The subunit composition, however, has evolved to respond properly to physiological signals.

Asunto(s)

ARN Polimerasa II/química , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/enzimología , Factores Asociados con la Proteína de Unión a TATA , Factor de Transcripción TFIID , Factores de Transcripción TFII , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/enzimología , Cromatografía de Afinidad , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Fosforilación , Estructura Cuaternaria de Proteína , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/enzimología , Schizosaccharomyces/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factor de Transcripción TFIIH , Factores de Transcripción/metabolismo

RESUMEN

The form of RNA polymerase II (RNAPII) engaged in transcriptional elongation was isolated. Elongating RNAPII was associated with a novel multisubunit complex, termed elongator, whose stable interaction was dependent on a hyperphosphorylated state of the RNAPII carboxy-terminal domain (CTD). A free form of elongator was also isolated, demonstrating the discrete nature of the complex, and free elongator could bind directly to RNAPII. The gene encoding the largest subunit of elongator, ELP1, was cloned. Phenotypes of yeast elp1 delta cells demonstrated an involvement of elongator in transcriptional elongation as well as activation in vivo. Our data indicate that the transition from transcriptional initiation to elongation involves an exchange of the multiprotein mediator complex for elongator in a reaction coupled to CTD hyperphosphorylation.

Asunto(s)

ARN Polimerasa II/genética , Saccharomyces cerevisiae/genética , Factores Generales de Transcripción , Factores de Transcripción/genética , Transcripción Genética/genética , Factores de Elongación Transcripcional , División Celular/genética , Clonación Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Regulación de la Expresión Génica/genética , Fenotipo , Fosforilación , Unión Proteica/genética , ARN Polimerasa II/química , Factores de Transcripción/química , Activación Transcripcional/genética

RESUMEN

Single particles of the mediator of transcriptional regulation (Mediator) and of RNA polymerase II holoenzyme were revealed by electron microscopy and image processing. Mediator alone appeared compact, but at high pH or in the presence of RNA polymerase II it displayed an extended conformation. Holoenzyme contained Mediator in a fully extended state, partially enveloping the globular polymerase, with points of apparent contact in the vicinity of the polymerase carboxyl-terminal domain and the DNA-binding channel. A similarity in appearance and conformational behavior of yeast and murine complexes indicates a conservation of Mediator structure among eukaryotes.

Asunto(s)

ARN Polimerasas Dirigidas por ADN/química , Proteínas Fúngicas/química , Conformación Proteica , Transactivadores/química , Factores de Transcripción/química , Animales , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas Fúngicas/metabolismo , Holoenzimas/química , Holoenzimas/metabolismo , Concentración de Iones de Hidrógeno , Ratones , Microscopía Electrónica , Pliegue de Proteína , Temperatura , Transactivadores/metabolismo , Factores de Transcripción/metabolismo

RESUMEN

Deletion of any one of three subunits of the yeast Mediator of transcriptional regulation, Med2, Pgd1 (Hrs1), and Sin4, abolished activation by Gal4-VP16 in vitro. By contrast, other Mediator functions, stimulation of basal transcription and of TFIIH kinase activity, were unaffected. A different but overlapping Mediator subunit dependence was found for activation by Gcn4. The genetic requirements for activation in vivo were closely coincident with those in vitro. A whole genome expression profile of a Deltamed2 strain showed diminished transcription of a subset of inducible genes but only minor effects on "basal" transcription. These findings make an important connection between transcriptional activation in vitro and in vivo, and identify Mediator as a "global" transcriptional coactivator.

Asunto(s)

Proteínas de Unión al ADN , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae , Transactivadores/metabolismo , Transcripción Genética , Activación Transcripcional , Sistema Libre de Células , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Modelos Genéticos , Mutación , Proteínas Quinasas/metabolismo , Relación Estructura-Actividad , Transactivadores/genética , Levaduras/genética

RESUMEN

Mediator was isolated from yeast on the basis of its requirement for transcriptional activation in a fully defined system. We have now identified three new members of mediator in the low molecular mass range by peptide sequence determination. These are the products of the NUT2, CSE2, and MED11 genes. The product of the NUT1 gene is evidently a component of mediator as well. NUT1 and NUT2 were earlier identified as negative regulators of the HO promoter, whereas mutations in CSE2 affect chromosome segregation. MED11 is a previously uncharacterized gene. The existence of these proteins in the mediator complex was verified by copurification and co-immunoprecipitation with RNA polymerase II holoenzyme.

Asunto(s)

ARN Polimerasa II/química , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Factores de Transcripción , Secuencia de Aminoácidos , Proteínas Fúngicas/análisis , Proteínas Fúngicas/genética , Holoenzimas/química , Complejo Mediador , Datos de Secuencia Molecular , Proteínas Nucleares/análisis , Proteínas Nucleares/genética , Pruebas de Precipitina , Unión Proteica , Saccharomyces cerevisiae/genética , Análisis de Secuencia , Homología de Secuencia de Aminoácido

RESUMEN

Mediator was resolved from yeast as a multiprotein complex on the basis of its requirement for transcriptional activation in a fully defined system. Three groups of mediator polypeptides could be distinguished: the products of five SRB genes, identified as suppressors of carboxy-terminal domain (CTD)-truncation mutants; products of four genes identified as global repressors; and six members of a new protein family, termed Med, thought to be primarily responsible for transcriptional activation. Notably absent from the purified mediator were Srbs 8, 9, 10, and 11, as well as members of the SWI/SNF complex. The CTD was required for function of mediator in vitro, in keeping with previous indications of involvement of the CTD in transcriptional activation in vivo. Evidence for human homologs of several mediator proteins, including Med7, points to similar mechanisms in higher cells.

Asunto(s)

Proteínas Fúngicas/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , ADN Complementario , Proteínas Fúngicas/genética , Humanos , Sustancias Macromoleculares , Complejo Mediador , Datos de Secuencia Molecular , Complejos Multiproteicos , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Transactivadores/genética , Factores de Transcripción/genética

RESUMEN

Protocols are presented for the preparation of a fully defined yeast RNA polymerase II transcription system, consisting of essentially pure TFIIB, -E, -F, and -H, TATA-binding protein, RNA polymerase II, and mediator of transcriptional regulation. This system, comprising 44 polypeptides, is able to initiate transcription at any of a dozen yeast and mammalian promoters thus far tested and responds to a variety of transcriptional activator proteins.

Asunto(s)

ARN Polimerasa II/genética , Proteínas de Saccharomyces cerevisiae , Factores Asociados con la Proteína de Unión a TATA , Factor de Transcripción TFIID , Factores de Transcripción TFII , Transcripción Genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Fosfoproteínas/química , Fosfoproteínas/genética , ARN Polimerasa II/química , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteína de Unión a TATA-Box , Factor de Transcripción TFIIB , Factor de Transcripción TFIIH , Factores de Transcripción/química , Factores de Transcripción/genética

RESUMEN

Yeast Rox3 protein, implicated by genetic evidence in both negative and positive transcriptional regulation, is identified as a mediator subunit by peptide sequence determination and is shown to copurify and co-immunoprecipitate with RNA polymerase II holoenzyme.

Asunto(s)

Proteínas Fúngicas/química , ARN Polimerasa II/química , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/química , Secuencia de Aminoácidos , Regulación Fúngica de la Expresión Génica , Sustancias Macromoleculares , Complejo Mediador , Datos de Secuencia Molecular , Pruebas de Precipitina , Saccharomyces cerevisiae/química

RESUMEN

The herpes simplex virus type 1 (HSV-1) origin binding protein, OBP, is a DNA helicase specifically stimulated by the viral single strand DNA-binding protein, ICP-8. The stimulation is dependent on direct protein-protein interactions between the C-terminal domain of OBP, delta OBP, and ICP 8 (Boehmer, P.E., Craigie, M.C., Stow, N.D., and Lehman, I.R. (1994) J. Biol. Chem. 269, 29329-29334). We have now observed that this interaction is dramatically influenced by the nature of the DNA ligand. Stable complexes between delta OBP, ICP 8, and double-stranded DNA, presented either as a specific duplex oligonucleotide or a restriction fragment containing the HSV-1 origin of replication, oriS, can be detected by gel chromatography and gel electrophoresis. In contrast, a single-stranded oligonucleotide, oligo(dT)65, will completely disrupt the complex between delta OBP and ICP 8. We therefore suggest that the interaction between delta OBP and ICP 8 serves to position the single strand DNA-binding protein with high precision onto single-stranded DNA at a replication fork or at an origin of DNA replication.

Asunto(s)

ADN Helicasas/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Virales/metabolismo , Secuencia de Bases , Sitios de Unión , Replicación del ADN , Ligandos , Datos de Secuencia Molecular

RESUMEN

The origin-binding protein (OBP) from herpes simplex virus 1 is a member of the SF2 helicase superfamily and is required for the initiation of DNA synthesis from a viral origin of DNA replication (oriS). The high-affinity binding sites for OBP in oriS, boxes I and II, are separated by an A+T-rich spacer. We used the gel retardation technique to examine the influence of this spacer sequence on the formation of a specific complex, referred to as complex II, between OBP and oriS. The formation of this OBP-oriS complex was greatly promoted by adenosine 5'-[gamma-thio]triphosphate and other nucleotide cofactors. Surprisingly, oriS constructs where the spacer sequence had been altered with approximately half of a helical turn (+4 or -6 base pairs) supported the formation of a more stable complex II than the wild-type origin. DNase I footprinting experiments showed that the cooperative binding of OBP to boxes I and II was affected by the length of the spacer sequence in the same way. In contrast, the ability of oriS-containing plasmids to replicate was most efficient with wild-type oriS. This paradox can be resolved if it is assumed that an ATP-dependent cooperative binding of OBP to properly spaced recognition sequences in oriS is required to induce a conformational change of DNA, thereby facilitating initiation of DNA replication.

Asunto(s)

Adenosina Trifosfato/metabolismo , Replicación del ADN , ADN Viral/biosíntesis , Proteínas de Unión al ADN/metabolismo , Nucleoproteínas/metabolismo , Simplexvirus/genética , Proteínas Virales/metabolismo , Animales , Secuencia de Bases , Línea Celular , Desoxirribonucleasa I , Datos de Secuencia Molecular , Estructura Molecular , Mariposas Nocturnas , Conformación de Ácido Nucleico , Unión Proteica

RESUMEN

The origin binding protein (OBP) of herpes simplex virus type 1 is required to activate a viral origin of replication in vivo. We have used intact OBP as well as a truncated form of the protein expressed in Escherichia coli to investigate the protein-protein interactions, as well as the protein-DNA interactions involved in the formation of a nucleoprotein complex at a viral origin of replication (oriS) in vitro. The salient findings demonstrate that the N-terminal part of OBP is required for the cooperative binding of OBP to three sites (boxes I, II, and III) within oriS. A detailed model for the interaction of OBP with the viral origins of replication oriS and oriL is presented.

Asunto(s)

Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Simplexvirus/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Bovinos , Cromatografía en Gel , Clonación Molecular , ADN/metabolismo , ADN Viral/genética , ADN Viral/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Desoxirribonucleasa I , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Genes Virales , Modelos Estructurales , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Conformación Proteica , Simplexvirus/genética , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificación

RESUMEN

The origin binding protein (OBP) or herpes simplex virus 1 has been expressed in Escherichia coli and used to study the role of multiple OBP binding sites in the herpes simplex virus #1 origin of replication, oris. Our results showed that the sequence CGTTCGCACTT was required for the binding of OBP to duplex DNA with high affinity. The minimal oris contains three repeats of this sequence or close derivatives thereof. Filter binding experiments have demonstrated that specific binding occurs to two of these repeats, box I and box II. An investigation using the DNase I footprinting technique revealed that the binding of OBP to box I and box II was cooperative and led to the formation of a highly organized complex in which the entire oris sequence was induced. We observed furthermore that the AT-rich sequence of the oris dyad was readily accessible to macromolecules even in the OBP.oris complex. The DNase I cleavage pattern of this sequence was, however, altered radically, indicating that a significant conformational change had occurred. A tentative structural model for the OBP-oris interaction is discussed on the basis of these observations.

Asunto(s)

Replicación del ADN , ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Simplexvirus/metabolismo , Proteínas Virales/metabolismo , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Clonación Molecular , ADN Viral/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Humanos , Cinética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Plásmidos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Simplexvirus/genética , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificación