RESUMEN
Although MDM2 plays a major role in regulating the stability of the p53 tumor suppressor protein, other poorly understood MDM2-independent pathways also exist. Human adenoviruses have evolved strategies to regulate p53 function and stability to permit efficient viral replication. One mechanism involves adenovirus E1B55K and E4orf6 proteins, which collaborate to target p53 for degradation. To determine the mechanism of this process, a multiprotein E4orf6-associated complex was purified and shown to contain a novel Cullin-containing E3 ubiquitin ligase that is (1) composed of Cullin family member Cul5, Elongins B and C, and the RING-H2 finger protein Rbx1(ROC1); (2) remarkably similar to the von Hippel-Lindau tumor suppressor and SCF (Skp1-Cul1/Cdc53-F-box) E3 ubiquitin ligase complexes; and (3) capable of stimulating ubiquitination of p53 in vitro in the presence of E1/E2 ubiquitin-activating and -conjugating enzymes. Cullins are activated by NEDD8 modification; therefore, to determine whether Cullin complexes are required for adenovirus-induced p53 degradation, studies were conducted in ts41 Chinese hamster ovary cells that are temperature sensitive for the NEDD8 pathway. E4orf6/E1B55K failed to induce the degradation of p53 at the nonpermissive temperature. Thus, our results identify a novel role for the Cullin-based machinery in regulation of p53.
Asunto(s)
Proteínas E1B de Adenovirus/metabolismo , Proteínas E4 de Adenovirus/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas E1B de Adenovirus/química , Animales , Western Blotting , Células CHO , Proteínas Portadoras/metabolismo , Línea Celular , Cricetinae , Elonguina , Humanos , Ligasas/química , Ligasas/metabolismo , Sustancias Macromoleculares , Ratones , Microscopía Confocal , Modelos Biológicos , Peso Molecular , Complejos Multiproteicos , Unión Proteica , Temperatura , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Ubiquitina/metabolismo , Ubiquitina-Proteína LigasasRESUMEN
The heterodimeric Elongin BC complex has been shown to interact in vitro and in mammalian cells with a conserved BC-box motif found in a growing number of proteins including RNA polymerase II elongation factor Elongin A, SOCS-box proteins, and the von Hippel-Lindau (VHL) tumor suppressor protein. Recently, the VHL-Elongin BC complex was found to interact with a module composed of Cullin family member Cul2 and RING-H2 finger protein Rbx1 to reconstitute a novel E3 ubiquitin ligase that activates ubiquitylation by the E2 ubiquitin-conjugating enzymes Ubc5 and Cdc34. In the context of the VHL ubiquitin ligase, Elongin BC functions as an adaptor that links the VHL protein to the Cul2/Rbx1 module, raising the possibility that the Elongin BC complex could function as an integral component of a larger family of E3 ubiquitin ligases by linking alternative BC-box proteins to Cullin/Rbx1 modules. In this report, we describe identification and purification from rat liver of a novel leucine-rich repeat-containing BC-box protein, MUF1, which we demonstrate is capable of assembling with a Cullin/Rbx1 module containing the Cullin family member Cul5 to reconstitute ubiquitin ligase activity. In addition, we show that the additional BC-box proteins Elongin A, SOCS1, and WSB1 are also capable of assembling with the Cul5/Rbx1 module to reconstitute potential ubiquitin ligases. Taken together, our findings identify MUF1 as a new member of the BC-box family of proteins, and they predict the existence of a larger family of Elongin BC-based E3 ubiquitin ligases.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Leucina/química , Factores de Transcripción/química , Complejos de Ubiquitina-Proteína Ligasa , Secuencia de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Animales , Proteínas Portadoras/aislamiento & purificación , Línea Celular , Clonación Molecular , ADN Complementario/metabolismo , Elonguina , Insectos , Ligasas/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/metabolismo , Secuencias Repetitivas de Aminoácido , Homología de Secuencia de Aminoácido , Factores de Transcripción/metabolismo , Enzimas Ubiquitina-Conjugadoras , Ubiquitina-Proteína Ligasas , Ubiquitinas/metabolismoRESUMEN
TFIIH is a multifunctional RNA polymerase II general initiation factor that includes two DNA helicases encoded by the Xeroderma pigmentosum complementation group B (XPB) and D (XPD) genes and a cyclin-dependent protein kinase encoded by the CDK7 gene. Previous studies have shown that the TFIIH XPB DNA helicase plays critical roles not only in transcription initiation, where it catalyzes ATP-dependent formation of the open complex, but also in efficient promoter escape, where it suppresses arrest of very early RNA polymerase II elongation intermediates. In this report, we present evidence that ATP-dependent TFIIH action in transcription initiation and promoter escape requires distinct regions of the DNA template; these regions are well separated from the promoter region unwound by the XPB DNA helicase and extend, respectively, approximately 23-39 and approximately 39-50 bp downstream from the transcriptional start site. Taken together, our findings bring to light a role for promoter DNA in TFIIH action and are consistent with the model that TFIIH translocates along promoter DNA ahead of the RNA polymerase II elongation complex until polymerase has escaped the promoter.
Asunto(s)
ADN/genética , Regiones Promotoras Genéticas , Factores de Transcripción TFII , Factores de Transcripción/fisiología , Transcripción Genética/fisiología , Secuencia de Bases , Datos de Secuencia Molecular , ARN Polimerasa II/metabolismo , Factor de Transcripción TFIIHRESUMEN
Recently, key advances in biochemical and structural studies of RNA polymerase II (pol II) and the basal transcriptional machinery have shed considerable light on the basic mechanisms underlying the initiation stage of eukaryotic mRNA synthesis. The development of methods for obtaining crystal structures of pol II and its complexes has revolutionized transcriptional studies and holds promise that aspects of initiation will soon be understood at atomic resolution; crosslinking studies have revealed intriguing features of the topology of the pol II initiation complex and provided working models for dynamic steps of initiation; and mechanistic studies have identified promoter escape as a critical step during initiation and brought to light novel roles for the general initiation factors TFIIE, TFIIF, and TFIIH in this process.
Asunto(s)
ADN Helicasas , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Transcripción Genética , Adenosina Trifosfatasas , Regulación de la Expresión Génica , Humanos , Factores de Transcripción/metabolismoRESUMEN
TFIIF, ELL, and Elongin belong to a class of RNA polymerase II transcription factors that function similarly to activate the rate of elongation by suppressing transient pausing by polymerase at many sites along DNA templates. SII is a functionally distinct RNA polymerase II elongation factor that promotes elongation by reactivating arrested polymerase. Studies of the mechanism of SII action have shown (i) that arrest of RNA polymerase II results from irreversible displacement of the 3'-end of the nascent transcript from the polymerase catalytic site and (ii) that SII reactivates arrested polymerase by inducing endonucleolytic cleavage of the nascent transcript by the polymerase catalytic site thereby creating a new transcript 3'-end that is properly aligned with the catalytic site and can be extended. SII also induces nascent transcript cleavage by paused but non-arrested RNA polymerase II elongation intermediates, leading to the proposal that pausing may result from reversible displacement of the 3'-end of nascent transcripts from the polymerase catalytic site. On the basis of evidence consistent with the model that TFIIF, ELL, and Elongin suppress pausing by preventing displacement of the 3'-end of the nascent transcript from the polymerase catalytic site, we investigated the possibility of cross-talk between SII and transcription factors TFIIF, ELL, and Elongin. These studies led to the discovery that TFIIF, ELL, and Elongin are all capable of inhibiting SII-induced nascent transcript cleavage by non-arrested RNA polymerase II elongation intermediates. Here we present these findings, which bring to light a novel activity associated with TFIIF, ELL, and Elongin and suggest that these transcription factors may expedite elongation not only by increasing the forward rate of nucleotide addition by RNA polymerase II, but also by inhibiting SII-induced nascent transcript cleavage by non-arrested RNA polymerase II elongation intermediates.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas de Neoplasias , Factores de Elongación de Péptidos , ARN Mensajero/metabolismo , Factores Generales de Transcripción , Factores de Transcripción TFII , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Factores de Elongación Transcripcional , Secuencia de Bases , ADN , Elonguina , Humanos , Hidrólisis , Regiones Promotoras Genéticas , Proteínas Recombinantes/metabolismoRESUMEN
Inherited mutations of the TFIIH helicase subunits xeroderma pigmentosum (XP) B or XPD yield overlapping DNA repair and transcription syndromes. The high risk of cancer in these patients is not fully explained by the repair defect. The transcription defect is subtle and has proven more difficult to evaluate. Here, XPB and XPD mutations are shown to block transcription activation by the FUSE Binding Protein (FBP), a regulator of c-myc expression, and repression by the FBP Interacting Repressor (FIR). Through TFIIH, FBP facilitates transcription until promoter escape, whereas after initiation, FIR uses TFIIH to delay promoter escape. Mutations in TFIIH that impair regulation by FBP and FIR affect proper regulation of c-myc expression and have implications in the development of malignancy.
Asunto(s)
Factores de Transcripción TFII , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Xerodermia Pigmentosa/metabolismo , Western Blotting , Línea Celular , ADN Helicasas/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Genes Dominantes , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/metabolismo , Mutación , Neoplasias/metabolismo , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Empalme de ARN , Proteínas de Unión al ARN , Proteínas Recombinantes/metabolismo , Proteínas Represoras/metabolismo , Factor de Transcripción TFIIH , Transcripción Genética , Transfección , Xerodermia Pigmentosa/genéticaRESUMEN
RNA polymerase II elongation factor ELL was recently purified from rat liver as a component of a multiprotein complex containing ELL and three ELL-associated proteins (EAPs) of approximately 45 (EAP45), approximately 30 (EAP30), and approximately 20 (EAP20) kDa (Shilatifard, A. (1998) J. Biol. Chem. 273, 11212-11217). Cloning of cDNA encoding the EAP30 protein revealed that it shares significant sequence similarity with the product of the Saccharomyces cerevisiae SNF8 gene (Schmidt, A. E., Miller, T., Schmidt, S. L., Shiekhattar, R., and Shilatifard, A. (1999) J. Biol. Chem. 274, 21981-21985), which is required for efficient derepression of glucose-repressed genes. Here we report the cloning of cDNAs encoding the EAP45 and EAP20 proteins. In addition, we identify the S. cerevisiae VPS36 and YJR102c genes as potential orthologs of EAP45 and EAP20 and show that they are previously uncharacterized SNF genes with properties very similar to SNF8.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica/fisiología , Glucosa/farmacología , Proteínas de Neoplasias , Factores de Elongación de Péptidos , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Clonación Molecular , Secuencia Conservada , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Drosophila melanogaster/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Humanos , Hígado/enzimología , Mamíferos , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Subunidades de Proteína , ARN Polimerasa II/metabolismo , Ratas , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Elongación TranscripcionalRESUMEN
Mutations in the VHL tumor suppressor gene result in constitutive expression of many hypoxia-inducible genes, at least in part because of increases in the cellular level of hypoxia-inducible transcription factor HIF1alpha, which in normal cells is rapidly ubiquitinated and degraded by the proteasome under normoxic conditions. The recent observation that the VHL protein is a subunit of an Skp1-Cul1/Cdc53-F-box (SCF)-like E3 ubiquitin ligase raised the possibility that VHL may be directly responsible for regulating cellular levels of HIF1alpha by targeting it for ubiquitination and proteolysis. In this report, we test this hypothesis directly. We report development of methods for production of the purified recombinant VHL complex and present direct biochemical evidence that it can function with an E1 ubiquitin-activating enzyme and E2 ubiquitin-conjugating enzyme to activate HIF1alpha ubiquitination in vitro. Our findings provide new insight into the function of the VHL tumor suppressor protein, and they provide a foundation for future investigations of the mechanisms underlying VHL regulation of oxygen-dependent gene expression.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Ligasas , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Factores de Transcripción , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Ubiquitinas/metabolismo , Animales , Baculoviridae/genética , Línea Celular , Genes Supresores de Tumor , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Proteínas/genética , Proteínas Recombinantes/metabolismo , Spodoptera , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauRESUMEN
The elongation stage of eukaryotic mRNA synthesis can be regulated by transcription factors that interact directly with the RNA polymerase II (pol II) elongation complex and by activities that modulate the structure of its chromatin template. Recent studies have revealed new elongation factors and have implicated the general initiation factors TFIIE, TFIIF and TFIIH, as well as the C-terminal domain (CTD) of the largest subunit of pol II, in elongation. The recently reported high-resolution crystal structure of RNA polymerase II, which provides insight into the architecture of the elongation complex, marks a new era of investigation into transcription elongation.
Asunto(s)
ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , ARN Mensajero/biosíntesis , Cromatina/metabolismo , Regulación de la Expresión Génica , Modelos Biológicos , ARN Polimerasa II/genética , Factores de Transcripción/química , Factores de Transcripción/metabolismoAsunto(s)
ARN Polimerasa II/química , Saccharomyces cerevisiae/enzimología , Sitios de Unión , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , ADN de Hongos/química , ADN de Hongos/metabolismo , Modelos Moleculares , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , ARN Polimerasa II/metabolismo , ARN de Hongos/química , ARN de Hongos/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Moldes Genéticos , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Transcription factor IIIA (TFIIIA) activates 5S ribosomal RNA gene transcription in eukaryotes. The protein from vertebrates has nine contiguous Cys(2)His(2)zinc fingers which function in nucleic acid binding, and a C-terminal region involved in transcription activation. In order to identify protein partners for TFIIIA, yeast two-hybrid screens were performed using the C-terminal region of Xenopus TFIIIA as an attractor and a rat cDNA library as a source of potential partners. A cDNA clone was identified which produced a protein in yeast that interacted with Xenopus TFIIIA but not with yeast TFIIIA. This rat clone was sequenced and the primary structure of the human homolog (termed TFIIIA-intP for TFIIIA-interacting protein) was determined from expressed sequence tags. In vitro interaction of recombinant human TFIIIA-intP with recombinant Xenopus TFIIIA was demonstrated by immuno-precipitation of the complex using anti-TFIIIA-intP antibody. Interaction of rat TFIIIA with rat TFIIIA-intP was indicated by co-chromatography of the two proteins on DEAE-5PW following fractionation of a rat liver extract on cation, anion and gel filtration resins. In a HeLa cell nuclear extract, recombinant TFIIIA-intP was able to stimulate TFIIIA-dependent transcription of the Xenopus 5S ribosomal RNA gene but not TFIIIA-independent transcription of the human adenovirus VA RNA gene.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular , ADN Complementario/química , ADN Complementario/genética , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Extractos Hepáticos/química , Extractos Hepáticos/metabolismo , Proteínas de Transporte de Membrana , Datos de Secuencia Molecular , Unión Proteica , ARN Ribosómico 5S/genética , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factor de Transcripción TFIIIA , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Transcripción Genética , Técnicas del Sistema de Dos Híbridos , XenopusRESUMEN
Elongin is a transcription elongation factor that was first identified in mammalian systems and is composed of the three subunits, elongin A, B, and C. Sequence homologues of elongin A and elongin C, but not elongin B, were identified in the yeast genome. Neither yeast elongin A nor C sequence homologues was required for cell viability. The two gene products could be purified from yeast as a complex. A recombinant form of the complex, which could only be produced in bacteria if the gene products were co-expressed, was purified over several chromatographic steps. The complex did not stimulate transcription elongation by yeast RNA polymerase II. Using limited proteolysis, the N-terminal 144 residues of yeast elongin A were shown to be sufficient for interaction with yeast elongin C. The purified complex of yeast elongin C/elongin A(1-143) was analyzed using circular dichroism and nuclear magnetic spectroscopy. These studies revealed that yeast elongin A is unfolded but undergoes a dramatic modification of its structure in the presence of elongin C, and that elongin C forms a stable dimer in the absence of elongin A.
Asunto(s)
Proteínas Fúngicas/química , Saccharomyces cerevisiae/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Dicroismo Circular , Dimerización , Elonguina , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Factores de Transcripción/aislamiento & purificación , Transcripción GenéticaRESUMEN
The RING-H2 finger protein Rbx1 is a subunit of the related SCF (Skp1-Cdc53/Cul1-F-box protein) and von Hippel-Lindau (VHL) tumor suppressor (elongin BC-Cul2-VHL) E3 ubiquitin ligase complexes, where it functions as a component of Cdc53/Rbx1 and Cul2/Rbx1 modules that activate ubiquitination of target proteins by the E2 ubiquitin-conjugating enzymes Cdc34 and Ubc5. Here we demonstrate that the Cdc53/Rbx1 and Cul2/Rbx1 modules also activate conjugation of the ubiquitin-like protein Rub1 to Cdc53 and Cul2 by the dedicated E2 Rub1 conjugating enzyme Ubc12. Our findings identify Rbx1 as a common component of enzyme systems responsible for ubiquitin and Rub1 modification of target proteins.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Proteínas de Ciclo Celular/metabolismo , Proteínas Cullin , Proteínas Fúngicas/metabolismo , Ligasas , Péptido Sintasas/fisiología , Proteínas/fisiología , Proteínas de Saccharomyces cerevisiae , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Secuencia de Aminoácidos , Proteínas Fúngicas/fisiología , Sustancias Macromoleculares , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Ligasas SKP Cullina F-box , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Ubiquitinas , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau , Zinc/metabolismoRESUMEN
The von Hippel-Lindau tumor suppressor protein (pVHL) negatively regulates hypoxia-inducible mRNAs such as the mRNA encoding vascular endothelial growth factor (VEGF). This activity has been linked to its ability to form multimeric complexes that contain elongin C, elongin B, and Cul2. To understand this process in greater detail, we performed a series of in vitro binding assays using pVHL, elongin B, and elongin C variants as well as synthetic peptide competitors derived from pVHL or elongin C. A subdomain of elongin C (residues 17-50) was necessary and sufficient for detectable binding to elongin B. In contrast, elongin B residues required for binding to elongin C were not confined to a discrete colinear domain. We found that the pVHL (residues 157-171) is necessary and sufficient for binding to elongin C in vitro and is frequently mutated in families with VHL disease. These mutations preferentially involve residues that directly bind to elongin C and/or alter the conformation of pVHL such that binding to elongin C is at least partially diminished. These results are consistent with the view that diminished binding of pVHL to the elongins plays a causal role in VHL disease.
Asunto(s)
Ligasas , Fragmentos de Péptidos/química , Proteínas/química , Factores de Transcripción/química , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Secuencia de Aminoácidos , Hipoxia de la Célula , Línea Celular , Elonguina , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica , Conformación Proteica , Proteínas/genética , Factores de Transcripción/genética , Transcripción Genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau , Enfermedad de von Hippel-Lindau/etiologíaRESUMEN
Transcription factor (TF) IIF is a multifunctional RNA polymerase II transcription factor that has well established roles in both transcription initiation, where it functions as a component of the preinitiation complex and is required for formation of the open complex and synthesis of the first phosphodiester bond of nascent transcripts, and in transcription elongation, where it is capable of interacting directly with the ternary elongation complex and stimulating the rate of transcription. In this report, we present evidence that TFIIF is also required for efficient promoter escape by RNA polymerase II. Our findings argue that TFIIF performs dual roles in this process. We observe (i) that TFIIF suppresses the frequency of abortive transcription by very early RNA polymerase II elongation intermediates by increasing their processivity and (ii) that TFIIF cooperates with TFIIH to prevent premature arrest of early elongation intermediates. In addition, our findings argue that two TFIIF functional domains mediate TFIIF action in promoter escape. First, we observe that a TFIIF mutant selectively lacking elongation activity supports TFIIH action in promoter escape, but is defective in suppressing the frequency of abortive transcription by very early RNA polymerase II elongation intermediates. Second, a TFIIF mutant selectively lacking initiation activity is more active than wild type TFIIF in increasing the processivity of very early elongation intermediates, but is defective in supporting TFIIH action in promoter escape. Taken together, our findings bring to light a function for TFIIF in promoter escape and support a role for TFIIF elongation activity in this process.
Asunto(s)
Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Factores de Transcripción TFII , Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Bases , Elonguina , Cinética , Modelos Genéticos , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Moldes Genéticos , Factor de Transcripción TFIIHRESUMEN
Rat pheochromocytoma (PC12) cells were stably transfected with either wild type or mutated human von Hippel-Lindau tumor suppressor protein (hpVHL). These proteins have opposing effects on regulating expression of the gene encoding tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. Whereas wild type hpVHL represses levels of TH mRNA and protein 5-fold, a truncated pVHL mutant, pVHL(1-115), induces accumulation of TH mRNA and protein 3-fold. hpVHL-induced inhibition of TH gene expression does not involve either a decrease in TH mRNA stability or repression of TH promoter activity. However, repression results from inhibition of RNA elongation at a downstream region of the TH gene. This elongation pause is accompanied by hpVHL sequestration in the nuclear extracts of elongins B and C, regulatory components of the transcription elongation heterotrimer SIII (elongin A/B/C). Hypoxia, a physiological stimulus for TH gene expression, alleviates the elongation block. A truncated pVHL mutant, pVHL(1-115), stimulates TH gene expression by increasing the efficiency of TH transcript elongation. This is the first report showing pVHL-dependent regulation of specific transcript elongation in vivo, as well as dominant negative activity of pVHL mutants in pheochromocytoma cells.
Asunto(s)
Hipoxia de la Célula , Ligasas , Proteínas/fisiología , ARN Mensajero/genética , Proteínas Supresoras de Tumor , Tirosina 3-Monooxigenasa/genética , Ubiquitina-Proteína Ligasas , Animales , Regulación hacia Abajo , Elonguina , Regulación de la Expresión Génica/genética , Genes Supresores de Tumor , Humanos , Células PC12 , Proteínas/genética , Proteínas/metabolismo , Ratas , Factores de Transcripción/metabolismo , Transfección , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauRESUMEN
Mutations of von Hippel-Lindau disease (VHL) tumor-suppressor gene product (pVHL) are found in patients with dominant inherited VHL syndrome and in the vast majority of sporadic clear cell renal carcinomas. The function of the pVHL protein has not been clarified. pVHL has been shown to form a complex with elongin B and elongin C (VBC) and with cullin (CUL)-2. In light of the structural analogy of VBC-CUL-2 to SKP1-CUL-1-F-box ubiquitin ligases, the ubiquitin ligase activity of VBC-CUL-2 was examined in this study. We show that VBC-CUL-2 exhibits ubiquitin ligase activity, and we identified UbcH5a, b, and c, but not CDC34, as the ubiquitin-conjugating enzymes of the VBC-CUL-2 ubiquitin ligase. The protein Rbx1/ROC1 enhances ligase activity of VBC-CUL-2 as it does in the SKP1-CUL-1-F-box protein ligase complex. We also found that pVHL associates with two proteins, p100 and p220, which migrate at a similar molecular weight as two major bands in the ubiquitination assay. Furthermore, naturally occurring pVHL missense mutations, including mutants capable of forming a complex with elongin B-elongin C-CUL-2, fail to associate with p100 and p220 and cannot exhibit the E3 ligase activity. These results suggest that pVHL might be the substrate recognition subunit of the VBC-CUL-2 E3 ligase. This is also, to our knowledge, the first example of a human tumor-suppressor protein being directly involved in the ubiquitin conjugation system which leads to the targeted degradation of substrate proteins.
Asunto(s)
Genes Supresores de Tumor , Ligasas/metabolismo , Proteínas/metabolismo , Proteínas Supresoras de Tumor , Proteínas Portadoras/metabolismo , Humanos , Peso Molecular , Mutación , Unión Proteica , Proteínas/genética , Proteínas Recombinantes/metabolismo , Células Tumorales Cultivadas , Ubiquitina-Proteína Ligasas , Ubiquitinas/química , Ubiquitinas/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauRESUMEN
Elongin is a heterotrimeric transcription elongation factor composed of subunits A, B, and C in mammals. Elongin A and C are F-box-containing and SKP1 homologue proteins, respectively, and are therefore of interest for their potential roles in cell cycle-dependent proteolysis. Mammalian elongin C interacts with both elongin A and elongin B, as well as with the von Hippel-Lindau tumor suppressor protein VHL. To investigate the corresponding interactions in yeast, we have utilized NMR spectroscopy combined with ultracentrifugal sedimentation experiments to examine complexes of yeast elongin C (Elc1) with yeast elongin A (Ela1) and two peptides from homologous regions of Ela1 and human VHL. Elc1 alone is a homotetramer composed of subunits with a structured N-terminal region and a dynamically unstable C-terminal region. Binding of a peptide fragment of the Elc1-interaction domain of Ela1 or with a homologous peptide from VHL promotes folding of the C-terminal region of Elc1 into two regular helical structures and dissociates Elc1 into homodimers. Moreover, analysis of the complex of Elc1 with the full Elc1-interaction domain of Ela1 reveals that the Elc1 homodimer is dissociated to preferentially form an Ela1/Elc1 heterodimer. Thus, elongin C is found to oligomerize in solution and to undergo significant structural rearrangements upon binding of two different partner proteins. These results suggest a structural basis for the interaction of an F-box-containing protein with a SKP1 homologue and the modulation of this interaction by the tumor suppressor VHL.
Asunto(s)
Ligasas , Proteínas/química , Proteínas/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Secuencia de Aminoácidos , Animales , Elonguina , Genes Supresores de Tumor , Humanos , Enlace de Hidrógeno , Sustancias Macromoleculares , Mamíferos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Ultracentrifugación , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauRESUMEN
Over the past few years, biochemical and genetic studies have shed considerable light on the structure and function of the RNA polymerase II (pol II) elongation complex and the transcription factors that control it. Novel elongation factors have been identified and their mechanisms of action characterized in increasing detail; new insights into the biological roles of elongation factors have been gained from genetic studies of the regulation of mRNA synthesis in yeast; and intriguing links between the pol II elongation machinery and the pathways of DNA repair and recombination have emerged.
Asunto(s)
Regulación de la Expresión Génica , ARN Polimerasa II/metabolismo , Transcripción Genética/genética , Animales , Humanos , Factores de Transcripción/metabolismoRESUMEN
TFIIH is an RNA polymerase II transcription factor that performs ATP-dependent functions in both transcription initiation, where it catalyzes formation of the open complex, and in promoter escape, where it suppresses arrest of the early elongation complex at promoter-proximal sites. TFIIH possesses three known ATP-dependent activities: a 3' --> 5' DNA helicase catalyzed by its XPB subunit, a 5' --> 3' DNA helicase catalyzed by its XPD subunit, and a carboxyl-terminal domain (CTD) kinase activity catalyzed by its CDK7 subunit. In this report, we exploit TFIIH mutants to investigate the contributions of TFIIH DNA helicase and CTD kinase activities to efficient promoter escape by RNA polymerase II in a minimal transcription system reconstituted with purified polymerase and general initiation factors. Our findings argue that the TFIIH XPB DNA helicase is primarily responsible for preventing premature arrest of early elongation intermediates during exit of polymerase from the promoter.