RESUMEN
Central to the development and survival of all organisms is the regulation of gene expression, which begins with the process of transcription catalyzed by RNA polymerases. During transcription of protein-coding genes, the general transcription factors (GTFs) work alongside RNA polymerase II (Pol II) to assemble the preinitiation complex at the transcription start site, open the promoter DNA, initiate synthesis of the nascent messenger RNA, transition to productive elongation, and ultimately terminate transcription. Through these different stages of transcription, Pol II is dynamically phosphorylated at the C-terminal tail of its largest subunit, serving as a control mechanism for Pol II elongation and a signaling/binding platform for co-transcriptional factors. The large number of core protein factors participating in the fundamental steps of transcription add dense layers of regulation that contribute to the complexity of temporal and spatial control of gene expression within any given cell type. The Pol II transcription system is highly conserved across different levels of eukaryotes; however, most of the information here will focus on the human Pol II system. This review walks through various stages of transcription, from preinitiation complex assembly to termination, highlighting the functions and mechanisms of the core machinery that participates in each stage.
Asunto(s)
ARN Polimerasa II , Transcripción Genética , Humanos , ARN Polimerasa II/química , Factores de Transcripción/metabolismo , Regiones Promotoras Genéticas , ARN MensajeroRESUMEN
The transcriptional activator p53 is a tumor suppressor protein that controls cellular pathways important for cell fate decisions, including cell cycle arrest, senescence, and apoptosis. It functions as a tetramer by binding to specific DNA sequences known as response elements (REs) to control transcription via interactions with co-regulatory complexes. Despite its biological importance, the mechanism by which p53 binds REs remains unclear. To address this, we have used an in vitro single molecule fluorescence approach to quantify the dynamic binding of full-length human p53 to five native REs in real time under equilibrium conditions. Our approach enabled us to quantify the oligomeric state of DNA-bound p53. We found little evidence that dimer/DNA complexes form as intermediates en route to binding or dissociation of p53 tetramer/DNA complexes. Interestingly, however, at some REs dimers can rapidly exchange from tetramer/DNA complexes. Real time kinetic measurements enabled us to determine rate constants for association and dissociation at all five REs, which revealed two kinetically distinct populations of tetrameric p53/RE complexes. For the less stable population, the rate constants for dissociation were larger at REs closest to consensus, showing that the more favorable binding sequences form the least kinetically stable complexes. Together our single molecule measurements provide new insight into mechanisms by which tetrameric p53 forms complexes on different native REs.
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Proteína p53 Supresora de Tumor , Proteínas Supresoras de Tumor , Humanos , Proteína p53 Supresora de Tumor/genética , Unión Proteica , Proteínas Supresoras de Tumor/genética , Elementos de Respuesta , ADN/metabolismoRESUMEN
Breast cancer is a leading cause of cancer-related deaths in women. Many genetic and behavioral risk factors can contribute to the initiation and progression of breast cancer, one being alcohol consumption. Numerous epidemiological studies have established a positive correlation between alcohol consumption and breast cancer; however, the molecular basis for this link remains ill defined. Elucidating ethanol-induced changes to global transcriptional programming in breast cells is important to ultimately understand how alcohol and breast cancer are connected mechanistically. We investigated induced transcriptional changes in response to a short cellular exposure to moderate levels of alcohol. We treated the nontumorigenic breast cell line MCF10A and the tumorigenic breast cell lines MDA-MB-231 and MCF7, with ethanol for 6 h, and then captured the changes to ongoing transcription using 4-thiouridine metabolic labeling followed by deep sequencing. Only the MCF10A cell line exhibited statistically significant changes in newly transcribed RNA in response to ethanol treatment. Further experiments revealed that some ethanol-upregulated genes are sensitive to the dose of alcohol treatment, while others are not. Gene Ontology and biochemical pathway analyses revealed that ethanol-upregulated genes in MCF10A cells are enriched in biological functions that could contribute to cancer development.
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Neoplasias de la Mama , Etanol , Femenino , Humanos , Etanol/efectos adversos , Mama , Neoplasias de la Mama/metabolismo , Línea CelularRESUMEN
Understanding how cells remember previous mechanical environments to influence their fate, or mechanical memory, informs the design of biomaterials and therapies in medicine. Current regeneration therapies, such as cartilage regeneration procedures, require 2D cell expansion processes to achieve large cell populations critical for the repair of damaged tissues. However, the limit of mechanical priming for cartilage regeneration procedures before inducing long-term mechanical memory following expansion processes is unknown, and mechanisms defining how physical environments influence the therapeutic potential of cells remain poorly understood. Here, we identify a threshold to mechanical priming separating reversible and irreversible effects of mechanical memory. After 16 population doublings in 2D culture, expression levels of tissue-identifying genes in primary cartilage cells (chondrocytes) are not recovered when transferred to 3D hydrogels, while expression levels of these genes were recovered for cells only expanded for eight population doublings. Additionally, we show that the loss and recovery of the chondrocyte phenotype correlates with a change in chromatin architecture, as shown by structural remodeling of the trimethylation of H3K9. Efforts to disrupt the chromatin architecture by suppressing or increasing levels of H3K9me3 reveal that only with increased levels of H3K9me3 did the chromatin architecture of the native chondrocyte phenotype partially return, along with increased levels of chondrogenic gene expression. These results further support the connection between the chondrocyte phenotype and chromatin architecture, and also reveal the therapeutic potential of inhibitors of epigenetic modifiers as disruptors of mechanical memory when large numbers of phenotypically suitable cells are required for regeneration procedures.
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Cartílago Articular , Cartílago , Condrocitos , Fenotipo , Cromatina/metabolismo , Epigénesis Genética , Diferenciación Celular , Ingeniería de Tejidos/métodosRESUMEN
Transcription is the critical first step in expressing a gene, during which an RNA polymerase (RNAP) synthesizes an RNA copy of one strand of the DNA that encodes a gene. Here we describe a laboratory experiment that uses a single assay to probe two important steps in transcription: (1) RNAP binding to DNA, and (2) the transcriptional activity of the polymerase. Students probe both these steps in a single experiment using a fluorescence-based electrophoretic mobility shift assay (EMSA) and commercially available Escherichia coli RNAP. As an inquiry-driven component, students add the transcriptional inhibitor rifampicin to reactions and draw conclusions about its mechanism of inhibition by determining whether it blocks polymerase binding to DNA or transcriptional activity. Depending on the curriculum and learning goals of individual courses, this experimental module could be easily expanded to include additional experimentation that mimics a research environment more closely. After completing the experiment students understand basic principles of transcription, mechanisms of inhibition, and the use of EMSAs to probe protein/DNA interactions.
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Proteínas de Unión al ADN , Escherichia coli , Humanos , Ensayo de Cambio de Movilidad Electroforética , Proteínas de Unión al ADN/química , Unión Proteica , Escherichia coli/genética , Escherichia coli/metabolismo , ADN/química , Transcripción GenéticaRESUMEN
Doxorubicin (Dox), a widely used anticancer DNA-binding drug, affects chromatin in multiple ways, and these effects contribute to both its efficacy and its dose-limiting side effects, especially cardiotoxicity. Here, we studied the effects of Dox on the chromatin binding of the architectural proteins high mobility group B1 (HMGB1) and the linker histone H1, and the transcription factor retinoic acid receptor (RARα) by fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) in live cells. At lower doses, Dox increased the binding of HMGB1 to DNA while decreasing the binding of the linker histone H1. At higher doses that correspond to the peak plasma concentrations achieved during chemotherapy, Dox reduced the binding of HMGB1 as well. This biphasic effect is interpreted in terms of a hierarchy of competition between the ligands involved and Dox-induced local conformational changes of nucleosome-free DNA. Combined, FRAP and FCS mobility data suggest that Dox decreases the overall binding of RARα to DNA, an effect that was only partially overcome by agonist binding. The intertwined interactions described are likely to contribute to both the effects and side effects of Dox.
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Proteína HMGB1 , Histonas , Cromatina , ADN , Doxorrubicina/farmacología , Proteína HMGB1/metabolismo , Histonas/metabolismo , Receptores de Ácido Retinoico/metabolismoRESUMEN
High Mobility Group Box (HMGB) proteins are small architectural DNA binding proteins that regulate multiple genomic processes such as DNA damage repair, nucleosome sliding, telomere homeostasis, and transcription. In doing so they control both normal cellular functions and impact a myriad of disease states, including cancers and autoimmune diseases. HMGB proteins bind to DNA and nucleosomes to modulate the local chromatin environment, which facilitates the binding of regulatory protein factors to the genome and modulates higher order chromosomal organization. Numerous studies over the years have characterized the structure and function of interactions between HMGB proteins and DNA, both biochemically and inside cells, providing valuable mechanistic insight as well as evidence these interactions influence pathological processes. This review highlights recent studies supporting the roles of HMGB1 and HMGB2 in global organization of the genome, as well as roles in transcriptional regulation and telomere maintenance via interactions with G-quadruplex structures. Moreover, emerging models for how HMGB proteins function as RNA binding proteins are presented. Nuclear HMGB proteins have broad regulatory potential to impact numerous aspects of cellular metabolism in normal and disease states.
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Proteínas HMGB/genética , Proteína HMGB1/genética , Proteína HMGB2/genética , Transcripción Genética , Cromatina/genética , Proteínas de Unión al ADN/genética , Genoma Humano/genética , Homeostasis , Humanos , Nucleosomas/genéticaRESUMEN
Infection by herpes simplex virus 1 (HSV-1) impacts nearly all steps of host cell gene expression. The regulatory mechanisms by which this occurs, and the interplay between host and viral factors, have yet to be fully elucidated. We investigated how the occupancy of RNA polymerase II (Pol II) on the host genome changes during HSV-1 infection and is impacted by the viral immediate early protein ICP4. Pol II ChIP-seq experiments revealed ICP4-dependent decreases and increases in Pol II levels across the bodies of hundreds of genes. Our data suggest ICP4 represses host transcription by inhibiting recruitment of Pol II and activates host genes by promoting release of Pol II from promoter proximal pausing into productive elongation. Consistent with this, ICP4 was required for the decrease in levels of the pausing factor NELF-A on several HSV-1-activated genes after infection. In the absence of infection, exogenous expression of ICP4 activated, but did not repress, transcription of some genes in a chromatin-dependent context. Our data support the model that ICP4 decreases promoter proximal pausing on host genes activated by infection and that ICP4 is necessary, but not sufficient, to repress transcription of host genes during viral infection.
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Herpes Simple/genética , Herpesvirus Humano 1/genética , Proteínas Inmediatas-Precoces/genética , Células HEK293 , Herpes Simple/metabolismo , Herpes Simple/virología , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/patogenicidad , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Regiones Promotoras Genéticas/genética , ARN Polimerasa II/metabolismo , Transcripción Genética/genéticaRESUMEN
The tumor suppressor protein p53 is critical for cell fate decisions, including apoptosis, senescence, and cell cycle arrest. p53 is a tetrameric transcription factor that binds DNA response elements to regulate transcription of target genes. p53 response elements consist of two decameric half-sites, and data suggest one p53 dimer in the tetramer binds to each half-site. Despite a broad literature describing p53 binding DNA, unanswered questions remain, due partly to the need for more quantitative and structural studies with full length protein. Here we describe a single molecule fluorescence system to visualize full length p53 tetramers binding DNA in real time. The data revealed a dynamic interaction in which tetrameric p53/DNA complexes assembled and disassembled without a dimer/DNA intermediate. On a wild type DNA containing two half sites, p53/DNA complexes existed in two kinetically distinct populations. p53 tetramers bound response elements containing only one half site to form a single population of complexes with reduced kinetic stability. Altering the spacing and helical phasing between two half sites affected both the population distribution of p53/DNA complexes and their kinetic stability. Our real time single molecule measurements of full length p53 tetramers binding DNA reveal the parameters that define the stability of p53/DNA complexes, and provide insight into the pathways by which those complexes assemble.
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Elementos de Respuesta , Proteína p53 Supresora de Tumor/metabolismo , Sitios de Unión , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , Unión ProteicaRESUMEN
RNA polymerase II (Pol II) and its general transcription factors assemble on the promoters of mRNA genes to form large macromolecular complexes that initiate transcription in a regulated manner. During early transcription, these complexes undergo dynamic rearrangement and disassembly as Pol II moves away from the start site of transcription and transitions into elongation. One step in disassembly is the release of the general transcription factor TFIIB, although the mechanism of release and its relationship to the activity of transcribing Pol II is not understood. We developed a single-molecule fluorescence transcription system to investigate TFIIB release in vitro. Leveraging our ability to distinguish active from inactive complexes, we found that nearly all transcriptionally active complexes release TFIIB during early transcription. Release is not dependent on the contacts TFIIB makes with its recognition element in promoter DNA. We identified two different points in early transcription at which release is triggered, reflecting heterogeneity across the population of actively transcribing complexes. TFIIB releases after both trigger points with similar kinetics, suggesting the rate of release is independent of the molecular transformations that prompt release. Together our data support the model that TFIIB release is important for Pol II to successfully escape the promoter as initiating complexes transition into elongation complexes.
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ADN/genética , ARN Polimerasa II/genética , Factor de Transcripción TFIIB/genética , Transcripción Genética/genética , Fluorescencia , Humanos , Cinética , Regiones Promotoras Genéticas/genética , ARN/biosíntesis , ARN/genética , Imagen Individual de Molécula/métodosRESUMEN
The muscle specific miRNA, miR-206, is important for the process of myogenesis; however, studying the function of miR-206 in muscle development and differentiation still proves challenging because the complement of mRNA targets it regulates remains undefined. In addition, miR-206 shares close sequence similarity to miR-1, another muscle specific miRNA, making it hard to study the impact of miR-206 alone in cell culture models. Here we used CRISPR/Cas9 technology to knockout miR-206 in C2C12 muscle cells. We show that knocking out miR-206 significantly impairs and delays differentiation and myotube formation, revealing that miR-206 alone is important for myogenesis. In addition, we use an experimental affinity purification technique to identify new mRNA targets of miR-206 in C2C12 cells. We identified over one hundred mRNAs as putative miR-206 targets. Functional experiments on six of these targets indicate that Adam19, Bgn, Cbx5, Smarce1, and Spg20 are direct miR-206 targets in C2C12 cells. Our data show a unique and important role for miR-206 in myogenesis.
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Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , MicroARNs/genética , Desarrollo de Músculos/genética , Interferencia de ARN , ARN Mensajero/genética , Regiones no Traducidas 3' , Animales , Sistemas CRISPR-Cas , Diferenciación Celular/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Mioblastos/metabolismoRESUMEN
Osteomyelitis remains a serious inflammatory bone disease that affects millions of individuals worldwide and for which there is no effective treatment. Despite scientific evidence that Staphylococcus bacteria are the most common causative species for human bacterial chondronecrosis with osteomyelitis (BCO), much remains to be understood about the underlying virulence mechanisms. Herein, we show increased levels of double-stranded RNA (dsRNA) in infected bone in a Staphylococcus-induced chicken BCO model and in human osteomyelitis samples. Administration of synthetic [poly(I:C)] or genetic (Alu) dsRNA induces human osteoblast cell death. Similarly, infection with Staphylococcus isolated from chicken BCO induces dsRNA accumulation and cell death in human osteoblast cell cultures. Both dsRNA administration and Staphylococcus infection activate NACHT, LRR and PYD domains-containing protein (NLRP)3 inflammasome and increase IL18 and IL1B gene expression in human osteoblasts. Pharmacologic inhibition with Ac-YVAD-cmk of caspase 1, a critical component of the NLRP3 inflammasome, prevents DICER1 dysregulation- and dsRNA-induced osteoblast cell death. NLRP3 inflammasome and its components are also activated in bone from BCO chickens and humans with osteomyelitis, compared with their healthy counterparts. These findings provide a rationale for the use of chicken BCO as a human-relevant spontaneous animal model for osteomyelitis and identify dsRNA as a new treatment target for this debilitating bone pathogenesis.
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Resorción Ósea/etiología , Osteoblastos/patología , Osteocondrosis/veterinaria , Osteomielitis/etiología , Enfermedades de las Aves de Corral/etiología , ARN Bicatenario/genética , Infecciones Estafilocócicas/complicaciones , Animales , Resorción Ósea/epidemiología , Resorción Ósea/patología , Pollos , Modelos Animales de Enfermedad , Humanos , Inflamasomas , Necrosis , Osteoblastos/metabolismo , Osteoblastos/microbiología , Osteocondrosis/epidemiología , Osteocondrosis/etiología , Osteomielitis/epidemiología , Osteomielitis/patología , Enfermedades de las Aves de Corral/epidemiología , Infecciones Estafilocócicas/microbiología , Staphylococcus/genética , Staphylococcus/aislamiento & purificaciónRESUMEN
RNA polymerase II (Pol II) transcribes eukaryotic mRNA genes. To initiate transcription, pre-initiation complexes (PICs) containing Pol II and general transcription factors (GTFs) form on the core promoters of target genes. In cells this process is regulated by transcriptional activators, co-activators, and chromatin modifying complexes. Reconstituted in vitro transcription systems are important tools for studying the enzymology and fundamental steps in the transcription reaction. In these systems, studying transcription can be complex due to the heterogeneous mixture of transcriptionally active and inactive complexes that assemble at promoters. Accordingly, we developed a technique to use single molecule microscopy to resolve this heterogeneity and distinguish transcriptionally active complexes from inactive complexes. This system uses fluorescently-labeled promoter DNA and a minimal reconstituted transcription system consisting of purified human Pol II and GTFs. Here we describe the materials, methods, and analysis required to study Pol II transcription at the single molecule level. The flexibility of our single molecule method allows for adaptation to answer diverse mechanistic questions about transcription that would otherwise be difficult to study using ensemble assays.
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Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Imagen Individual de Molécula/métodos , Transcripción Genética , Humanos , Imagen Óptica/métodosRESUMEN
Cellular transcriptional programs are tightly controlled but can profoundly change in response to environmental challenges or stress. Here we describe global changes in mammalian RNA polymerase II (Pol II) occupancy at mRNA genes in response to heat shock and after recovery from the stress. After a short heat shock, Pol II occupancy across thousands of genes decreased, consistent with widespread transcriptional repression, whereas Pol II occupancy increased at a small number of genes in a manner consistent with activation. Most striking, however, was loss of the Pol II peak near the 3' ends of mRNA genes, coupled to a gain in polymerase occupancy extending tens of kilobases downstream of 3' ends. Typical patterns of 3' end occupancy were largely restored 60 min after cells returned to normal growth temperatures. These changes in polymerase occupancy revealed a heat shock-induced loss of normal termination, which was potent, global, and reversible. The occupancy of the termination factor CPSF73 at the 3' ends of representative genes was reduced after heat shock, suggesting a mechanism for impaired termination. The data support a model in which heat shock induces widespread repression of transcriptional initiation and loss of transcription termination, which reverses as cells return to homeostasis.
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Respuesta al Choque Térmico/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , Terminación de la Transcripción Genética , Animales , Inmunoprecipitación de Cromatina , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Ratones , Modelos Genéticos , Células 3T3 NIHRESUMEN
PURPOSE: To assess the success rate, image quality, and the ability to stage liver fibrosis of a standard 2D gradient-recalled echo (GRE) and four different spin-echo (SE) magnetic resonance elastography (MRE) sequences in patients with different liver iron concentrations. MATERIALS AND METHODS: A total of 332 patients who underwent 3T MRE examinations that included liver fat and iron quantification were enrolled, including 136 patients with all five MRE techniques. Thirty-four patients had biopsy results for fibrosis staging. The liver stiffness, region of interest area, image quality, and success rate of the five sequences were compared in 115/136 patients. The area under the receiver operating characteristic curves (AUCs) and the accuracies for diagnosing early-stage fibrosis and advanced fibrosis were compared. The effect of BMI (body mass index), the R2* relaxation time, and fat fraction on the image quality and liver stiffness measurements were analyzed. RESULTS: The success rates were significantly higher in the four SE sequences (99.1-100%) compared with GRE MRE (85.3%) (all P < 0.001). There were significant differences of the mean ROI area between every pair of sequences (all P < 0.0001). There were no significant differences in the AUC of the five MRE sequences for discriminating advanced fibrosis (10 P-values ranging from 0.2410-0.9171). R2* had a significant effect on the success rate and image quality for the noniron 2D echo-planar imaging (EPI), 3D EPI and 2D GRE (all P < 0.001) sequences. BMI had a significant effect on the iron 2D EPI (P = 0.0230) and iron 2D SE (P = 0.0040) sequences. CONCLUSION: All five techniques showed good diagnostic performance in staging liver fibrosis. The SE MRE sequences had higher success rates and better image quality than GRE MRE in 3T clinical hepatic imaging. LEVEL OF EVIDENCE: 3 Technical Efficacy: Stage 5 J. Magn. Reson. Imaging 2018;47:976-987.
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Diagnóstico por Imagen de Elasticidad/métodos , Interpretación de Imagen Asistida por Computador/métodos , Cirrosis Hepática/diagnóstico por imagen , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Artefactos , Niño , Femenino , Humanos , Hígado/diagnóstico por imagen , Hígado/patología , Cirrosis Hepática/patología , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Estudios Retrospectivos , Adulto JovenRESUMEN
Transcription by RNA polymerase II (Pol II) is dictated in part by core promoter elements, which are DNA sequences flanking the transcription start site (TSS) that help direct the proper initiation of transcription. Taking advantage of recent advances in genome-wide sequencing approaches, Vo ngoc and colleagues (pp. 6-11) identified transcripts with focused sites of initiation and found that many were transcribed from promoters containing a new consensus sequence for the human initiator (Inr) core promoter element.
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Regiones Promotoras Genéticas , Sitio de Iniciación de la Transcripción , Secuencia de Bases , Secuencia de Consenso , Humanos , ARN Polimerasa II/genética , TATA Box , Transcripción GenéticaRESUMEN
High mobility group box protein 1 (HMGB1) is an architectural protein that facilitates the formation of protein-DNA assemblies involved in transcription, recombination, DNA repair, and chromatin remodeling. Important to its function is the ability of HMGB1 to bend DNA non-sequence specifically. HMGB1 contains two HMG boxes that bind and bend DNA (the A box and the B box) and a C-terminal acidic tail. We investigated how these domains contribute to DNA bending by HMGB1 using single-molecule fluorescence resonance energy transfer (FRET), which enabled us to resolve heterogeneous populations of bent and unbent DNA. We found that full-length (FL) HMGB1 bent DNA more than the individual A and B boxes. Removing the C-terminal tail resulted in a protein that bent DNA to a greater extent than the FL protein. These data suggest that the A and B boxes simultaneously bind DNA in the absence of the C-terminal tail, but the tail modulates DNA binding and bending by one of the HMG boxes in the FL protein. Indeed, a construct composed of the B box and the C-terminal tail only bent DNA at higher protein concentrations. Moreover, in the context of the FL protein, mutating the A box such that it could not bend DNA resulted in a protein that bent DNA similar to a single HMG box and only at higher protein concentrations. We propose a model in which the HMGB1 C-terminal tail serves as an intramolecular damper that modulates the interaction of the B box with DNA.
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ADN/metabolismo , Proteína HMGB1/metabolismo , Conformación de Ácido Nucleico , Transferencia Resonante de Energía de Fluorescencia , Unión Proteica , Dominios ProteicosRESUMEN
Transcription by RNA polymerase II (Pol II) is a complex process that requires general transcription factors and Pol II to assemble on DNA into preinitiation complexes that can begin RNA synthesis upon binding of NTPs (nucleoside triphosphate). The pathways by which preinitiation complexes form, and how this impacts transcriptional activity are not completely clear. To address these issues, we developed a single molecule system using TIRF (total internal reflection fluorescence) microscopy and purified human transcription factors, which allows us to visualize transcriptional activity at individual template molecules. We see that stable interactions between polymerase II (Pol II) and a heteroduplex DNA template do not depend on general transcription factors; however, transcriptional activity is highly dependent upon TATA-binding protein, TFIIB and TFIIF. We also found that subsets of general transcription factors and Pol II can form stable complexes that are precursors for functional transcription complexes upon addition of the remaining factors and DNA. Ultimately we found that Pol II, TATA-binding protein, TFIIB and TFIIF can form a quaternary complex in the absence of promoter DNA, indicating that a stable network of interactions exists between these proteins independent of promoter DNA. Single molecule studies can be used to learn how different modes of preinitiation complex assembly impact transcriptional activity.
Asunto(s)
Microscopía Fluorescente/métodos , ARN Polimerasa II/metabolismo , Imagen Individual de Molécula/métodos , Factores Generales de Transcripción/metabolismo , Iniciación de la Transcripción Genética , ADN/genética , ADN/metabolismo , Pruebas de Enzimas , Enzimas Inmovilizadas/metabolismo , Colorantes Fluorescentes , Humanos , Regiones Promotoras Genéticas , Estabilidad Proteica , Imagen Individual de Molécula/instrumentación , Moldes Genéticos , Factor de Transcripción TFIIB/metabolismo , Factores de Transcripción TFII/metabolismoRESUMEN
Transcription by RNA polymerase II (Pol II) is required to produce mRNAs and some noncoding RNAs (ncRNAs) within mammalian cells. This coordinated process is precisely regulated by multiple factors, including many recently discovered ncRNAs. In this perspective, we will discuss newly identified ncRNAs that facilitate DNA looping, regulate transcription factor binding, mediate promoter-proximal pausing of Pol II, and/or interact with Pol II to modulate transcription. Moreover, we will discuss new roles for ncRNAs, as well as a novel Pol II RNA-dependent RNA polymerase activity that regulates an ncRNA inhibitor of transcription. As the multifaceted nature of ncRNAs continues to be revealed, we believe that many more ncRNA species and functions will be discovered.