RESUMEN
The transition from transcription initiation to elongation is highly regulated in human cells but remains incompletely understood at the structural level. In particular, it is unclear how interactions between RNA polymerase II (RNA Pol II) and initiation factors are broken to enable promoter escape. Here, we reconstitute RNA Pol II promoter escape in vitro and determine high-resolution structures of initially transcribing complexes containing 8-, 10-, and 12-nt ordered RNAs and two elongation complexes containing 14-nt RNAs. We suggest that promoter escape occurs in three major steps. First, the growing RNA displaces the B-reader element of the initiation factor TFIIB without evicting TFIIB. Second, the rewinding of the transcription bubble coincides with the eviction of TFIIA, TFIIB, and TBP. Third, the binding of DSIF and NELF facilitates TFIIE and TFIIH dissociation, establishing the paused elongation complex. This three-step model for promoter escape fills a gap in our understanding of the initiation-elongation transition of RNA Pol II transcription.
Asunto(s)
Fosfoproteínas , Regiones Promotoras Genéticas , ARN Polimerasa II , Proteína de Unión a TATA-Box , Factor de Transcripción TFIIB , Factores de Transcripción , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Humanos , Factor de Transcripción TFIIB/metabolismo , Factor de Transcripción TFIIB/genética , Proteína de Unión a TATA-Box/metabolismo , Proteína de Unión a TATA-Box/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Iniciación de la Transcripción Genética , Factor de Transcripción TFIIH/metabolismo , Factor de Transcripción TFIIH/genética , Factor de Transcripción TFIIH/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Unión Proteica , Factor de Transcripción TFIIA/metabolismo , Factor de Transcripción TFIIA/genética , Transcripción Genética , Elongación de la Transcripción Genética , ARN/metabolismo , ARN/genética , Factores de Transcripción TFII/metabolismo , Factores de Transcripción TFII/genéticaRESUMEN
Although TFIIB is widely regarded as an initiation factor, recent reports have implicated it in multiple aspects of eukaryotic transcription. To investigate the broader role of TFIIB in transcription, we performed quantitative proteomic analysis of yeast TFIIB. We purified two different populations of TFIIB; one from soluble cell lysate, which is not engaged in transcription, and the other from the chromatin fraction which yields the transcriptionally active form of the protein. TFIIB purified from the chromatin exhibits several interactions that explain its non-canonical roles in transcription. RNAPII, TFIIF and TFIIH were the only components of the preinitiation complex with a significant presence in chromatin TFIIB. A notable feature was enrichment of all subunits of CF1 and Rat1 3' end processing-termination complexes in chromatin-TFIIB preparation. Subunits of the CPF termination complex were also detected in both chromatin and soluble derived TFIIB preparations. These results may explain the presence of TFIIB at the 3' end of genes during transcription as well as its role in promoter-termination interaction.
Asunto(s)
Mapas de Interacción de Proteínas , ARN Polimerasa II , Factor de Transcripción TFIIB/genética , Factor de Transcripción TFIIB/metabolismo , ARN Polimerasa II/genética , Proteómica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cromatina/genética , Cromatina/metabolismoRESUMEN
Transcription factor IIB (TFIIB) is a general transcription factor for RNA polymerase II, exerting its influence across various biological contexts. In the majority of eukaryotes, TFIIB typically has two homologs, serving as general transcription factors for RNA polymerase I and III. In plants, however, the TFIIB-related protein family has expanded greatly, with 14 and 9 members in Arabidopsis and rice, respectively. BRP5/pollen-expressed transcription factor 2 (PTF2) proteins belong to a subfamily of TFIIB-related proteins found only in plants and algae. The prior analysis of an Arabidopsis atbrp5 mutant, characterized by a T-DNA insertion at the 5' untranslated region, demonstrated the essential role of BRP5/PTF2 during the process of pollen germination and embryogenesis in Arabidopsis. Using a rice transformation system based on CRISPR/Cas9 technology, we have generated transgenic rice plants containing loss-of-function frameshift mutations in the BRP5/PTF2 gene. Unlike in the Arabidopsis atbrp5 mutant, the brp5/ptf2 frameshift mutations were not transmitted to progeny in rice, indicating an essential role of BRP5/PTF2 in both male and female gamete development or viability. The silencing of rice BRP5/PTF2 expression through RNA interference (RNAi) had little effect on vegetative growth and panicle formation but strongly affected pollen development and grain formation. Genetic analysis revealed that strong RNAi silencing of rice BRP5/PTF2 was still transmissible to progeny almost exclusively through female gametes, as found in the Arabidopsis atbrp5 knockdown mutant. Thus, reduced rice BRP5/PTF2 expression impacted pollen preferentially by interfering with male gamete development or viability. Drawing upon these findings, we posit that BRP5/PTF2 assumes a distinct and imperative function in the realm of plant sexual reproduction.
Asunto(s)
Oryza , Proteínas de Plantas , Factor de Transcripción TFIIB , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Gametogénesis , Regulación de la Expresión Génica de las Plantas , Oryza/genética , Oryza/metabolismo , Plantas/metabolismo , Polen/metabolismo , Factor de Transcripción TFIIB/metabolismo , Proteínas de Plantas/metabolismoRESUMEN
Eukaryotic RNA polymerase I (Pol I) products play fundamental roles in ribosomal assembly, protein synthesis, metabolism and cell growth. Abnormal expression of both Pol I transcription-related factors and Pol I products causes a range of diseases, including ribosomopathies and cancers. However, the factors and mechanisms governing Pol I-dependent transcription remain to be elucidated. Here, we report that transcription factor IIB-related factor 1 (BRF1), a subunit of transcription factor IIIB required for RNA polymerase III (Pol III)-mediated transcription, is a nucleolar protein and modulates Pol I-mediated transcription. We showed that BRF1 can be localized to the nucleolus in several human cell types. BRF1 expression correlates positively with Pol I product levels and tumour cell growth in vitro and in vivo. Pol III transcription inhibition assays confirmed that BRF1 modulates Pol I-directed transcription in an independent manner rather than through a Pol III product-to-45S pre-rRNA feedback mode. Mechanistically, BRF1 binds to the Pol I transcription machinery components and can be recruited to the rDNA promoter along with them. Additionally, alteration of BRF1 expression affects the recruitment of Pol I transcription machinery components to the rDNA promoter and the expression of TBP and TAF1A. These findings indicate that BRF1 modulates Pol I-directed transcription by controlling the expression of selective factor 1 subunits. In summary, we identified a novel role of BRF1 in Pol I-directed transcription, suggesting that BRF1 can independently regulate both Pol I- and Pol III-mediated transcription and act as a key coordinator of Pol I and Pol III.
Asunto(s)
Neoplasias , Factores Asociados con la Proteína de Unión a TATA , Humanos , ADN Ribosómico/genética , Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN Polimerasa I/genética , ARN Polimerasa I/metabolismo , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIIB/genética , Factor de Transcripción TFIIB/metabolismo , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The effects of rapid acute depletion of components of RNA polymerase II (Pol II) general transcription factors (GTFs) that are thought to be critical for formation of preinitiation complexes (PICs) and initiation in vitro were quantified in HAP1 cells using precision nuclear run-on sequencing (PRO-Seq). The average dependencies for each factor across >70 000 promoters varied widely even though levels of depletions were similar. Some of the effects could be attributed to the presence or absence of core promoter elements such as the upstream TBP-specificity motif or downstream G-rich sequences, but some dependencies anti-correlated with such sequences. While depletion of TBP had a large effect on most Pol III promoters only a small fraction of Pol II promoters were similarly affected. TFIIB depletion had the largest general effect on Pol II and also correlated with apparent termination defects downstream of genes. Our results demonstrate that promoter activity is combinatorially influenced by recruitment of TFIID and sequence-specific transcription factors. They also suggest that interaction of the preinitiation complex (PIC) with nucleosomes can affect activity and that recruitment of TFIID containing TBP only plays a positive role at a subset of promoters.
Asunto(s)
ARN Polimerasa II , Factores de Transcripción , Humanos , Factor de Transcripción TFIIB/genética , Factor de Transcripción TFIIB/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/metabolismo , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Transcripción Genética , TATA Box/genética , ARN Polimerasa III/genéticaRESUMEN
The initiator element is a core promoter element encompassing the transcription start site, which is found in yeast, Drosophila, and human promoters. This element is observed in TATA-less promoters. Several studies have defined transcription factor requirements and additional cofactors that are needed for transcription initiation of initiator-containing promoters. However, those studies have been performed with additional core promoters in addition to the initiator. In this work, we have defined the pathway of preinitiation complex formation on the fission yeast nmt1 gene promoter, which contains a functional initiator with striking similarity to the initiator of the human dihydrofolate reductase (hDHFR) gene and to the factor requirement for transcription initiation of the nmt1 gene promoter. The results show that the nmt1 gene promoter possesses an initiator encompassing the transcription start site, and several conserved base positions are required for initiator function. A preinitiation complex formation on the nmt1 initiator can be started by TBP/TFIIA or TBP/TFIIB, but not TBP alone, and afterwards follows the same pathway as preinitiation complex formation on TATA-containing promoters. Transcription initiation is dependent on the general transcription factors TBP, TFIIB, TFIIE, TFIIF, TFIIH, RNA polymerase II, Mediator, and a cofactor identified as transcription cofactor for initiator function (TCIF), which is a high-molecular-weight protein complex of around 500 kDa. However, the TAF subunits of TFIID were not required for the nmt1 initiator transcription, as far as we tested. We also demonstrate that other initiators of the nmt1/hDHFR family can be transcribed in fission yeast whole-cell extracts.
Asunto(s)
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Factor de Transcripción TFIIB/genética , Factor de Transcripción TFIIB/metabolismo , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/metabolismo , Transcripción GenéticaRESUMEN
The role of general transcription factor TFIIB in transcription extends well beyond its evolutionarily conserved function in initiation. Chromatin localization studies demonstrating binding of TFIIB to both the 5' and 3' ends of genes in a diverse set of eukaryotes strongly suggested a rather unexpected role of the factor in termination. TFIIB indeed plays a role in termination of transcription. TFIIB occupancy of the 3' end is possibly due to its interaction with the termination factors residing there. Interaction of the promoter-bound TFIIB with factors occupying the 3' end of a gene may be the basis of transcription-dependent gene looping. The proximity of the terminator-bound factors with the promoter in a gene loop has the potential to terminate promoter-initiated upstream anti-sense transcription thereby conferring promoter directionality. TFIIB, therefore, is emerging as a factor with pleiotropic roles in the transcription cycle. This could be the reason for preferential targeting of TFIIB by viruses. Further studies are needed to understand the critical role of TFIIB in viral pathogenesis in the context of its newly identified roles in termination, gene looping and promoter directionality.
Asunto(s)
Eucariontes , ARN Polimerasa II , Eucariontes/genética , Eucariontes/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , Factor de Transcripción TFIIB/genética , Factor de Transcripción TFIIB/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The TATA box-binding protein (TBP) is highly conserved throughout eukaryotes and plays a central role in the assembly of the transcription preinitiation complex (PIC) at gene promoters. TBP binds and bends DNA, and directs adjacent binding of the transcription factors TFIIA and TFIIB for PIC assembly. Here, we show that yeast TBP can bind to a nucleosome containing the Widom-601 sequence and that TBP-nucleosome binding is stabilized by TFIIA. We determine three cryo-electron microscopy (cryo-EM) structures of TBP-nucleosome complexes, two of them containing also TFIIA. TBP can bind to superhelical location (SHL) -6, which contains a TATA-like sequence, but also to SHL +2, which is GC-rich. Whereas binding to SHL -6 can occur in the absence of TFIIA, binding to SHL +2 is only observed in the presence of TFIIA and goes along with detachment of upstream terminal DNA from the histone octamer. TBP-nucleosome complexes are sterically incompatible with PIC assembly, explaining why a promoter nucleosome generally impairs transcription and must be moved before initiation can occur.
Asunto(s)
ADN/metabolismo , Nucleosomas/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIIA/metabolismo , Factor de Transcripción TFIIB/metabolismo , ADN/química , Modelos Moleculares , Nucleosomas/química , Unión Proteica , Conformación Proteica , Saccharomyces cerevisiae/metabolismo , Factor de Transcripción TFIIA/genética , Factor de Transcripción TFIIB/genéticaRESUMEN
Mediator is a conserved coactivator complex that enables the regulated initiation of transcription at eukaryotic genes1-3. Mediator is recruited by transcriptional activators and binds the pre-initiation complex (PIC) to stimulate the phosphorylation of RNA polymerase II (Pol II) and promoter escape1-6. Here we prepare a recombinant version of human Mediator, reconstitute a 50-subunit Mediator-PIC complex and determine the structure of the complex by cryo-electron microscopy. The head module of Mediator contacts the stalk of Pol II and the general transcription factors TFIIB and TFIIE, resembling the Mediator-PIC interactions observed in the corresponding complex in yeast7-9. The metazoan subunits MED27-MED30 associate with exposed regions in MED14 and MED17 to form the proximal part of the Mediator tail module that binds activators. Mediator positions the flexibly linked cyclin-dependent kinase (CDK)-activating kinase of the general transcription factor TFIIH near the linker to the C-terminal repeat domain of Pol II. The Mediator shoulder domain holds the CDK-activating kinase subunit CDK7, whereas the hook domain contacts a CDK7 element that flanks the kinase active site. The shoulder and hook domains reside in the Mediator head and middle modules, respectively, which can move relative to each other and may induce an active conformation of the CDK7 kinase to allosterically stimulate phosphorylation of the C-terminal domain.
Asunto(s)
Microscopía por Crioelectrón , Complejo Mediador/química , Complejo Mediador/ultraestructura , ARN Polimerasa II/química , ARN Polimerasa II/ultraestructura , Regulación Alostérica , Sitios de Unión , Dominio Catalítico , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/metabolismo , ADN Complementario/genética , Humanos , Complejo Mediador/metabolismo , Modelos Moleculares , Fosforilación , Unión Proteica , ARN Polimerasa II/metabolismo , Factor de Transcripción TFIIB/química , Factor de Transcripción TFIIB/metabolismo , Factores de Transcripción TFII/química , Factores de Transcripción TFII/metabolismo , Iniciación de la Transcripción Genética , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
The genome-wide architecture of chromatin-associated proteins that maintains chromosome integrity and gene regulation is not well defined. Here we use chromatin immunoprecipitation, exonuclease digestion and DNA sequencing (ChIP-exo/seq)1,2 to define this architecture in Saccharomyces cerevisiae. We identify 21 meta-assemblages consisting of roughly 400 different proteins that are related to DNA replication, centromeres, subtelomeres, transposons and transcription by RNA polymerase (Pol) I, II and III. Replication proteins engulf a nucleosome, centromeres lack a nucleosome, and repressive proteins encompass three nucleosomes at subtelomeric X-elements. We find that most promoters associated with Pol II evolved to lack a regulatory region, having only a core promoter. These constitutive promoters comprise a short nucleosome-free region (NFR) adjacent to a +1 nucleosome, which together bind the transcription-initiation factor TFIID to form a preinitiation complex. Positioned insulators protect core promoters from upstream events. A small fraction of promoters evolved an architecture for inducibility, whereby sequence-specific transcription factors (ssTFs) create a nucleosome-depleted region (NDR) that is distinct from an NFR. We describe structural interactions among ssTFs, their cognate cofactors and the genome. These interactions include the nucleosomal and transcriptional regulators RPD3-L, SAGA, NuA4, Tup1, Mediator and SWI-SNF. Surprisingly, we do not detect interactions between ssTFs and TFIID, suggesting that such interactions do not stably occur. Our model for gene induction involves ssTFs, cofactors and general factors such as TBP and TFIIB, but not TFIID. By contrast, constitutive transcription involves TFIID but not ssTFs engaged with their cofactors. From this, we define a highly integrated network of gene regulation by ssTFs.
Asunto(s)
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genoma Fúngico/genética , Complejos Multiproteicos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Coenzimas/metabolismo , Complejos Multiproteicos/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa I/metabolismo , ARN Polimerasa II/metabolismo , ARN Polimerasa III/metabolismo , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIIB/genética , Factor de Transcripción TFIIB/metabolismo , Factor de Transcripción TFIID , Factores de Transcripción/metabolismoRESUMEN
General transcription factors and core promoter elements play a pivotal role in RNA polymerase II (Pol II)-mediated transcription initiation. In the previous work, we have defined a TFIIA recognition element (IIARE) that modulates Pol II-directed gene transcription in a promoter context-dependent manner. However, how TFIIA interacts with the IIARE and whether the interaction between TFIIA and the IIARE is involved in the regulation of gene transcription by Pol II are not fully understood. In the present study, we confirm that both K348 and K350 residues in TFIIAαß are required for the interaction between TFIIAαß and the IIARE. Disruption of the interaction between them by gene mutations dampens TFIIAαß binding to the AdML-IIARE promoter and the transcriptional activation of the promoter containing a IIARE in vitro and in vivo. Stable expression of the TFIIAαß mutant containing both K348A and K350A in the cell line with endogenous TFIIAαß silence represses endogenous gene expression by reducing the occupancies of TFIIAαß, TBP, p300, and Pol II at the promoters containing a IIARE. The findings from this study provide a novel insight into the regulatory mechanism of gene transcription mediated by TFIIA and the IIARE.
Asunto(s)
Sitios de Unión , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Elementos de Respuesta , Factor de Transcripción TFIIA/metabolismo , Factor de Transcripción TFIIB/metabolismo , Activación Transcripcional , Aminoácidos , Secuencia de Bases , Línea Celular , Humanos , Modelos Moleculares , Mutación , Motivos de Nucleótidos , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Factor de Transcripción TFIIA/química , Factor de Transcripción TFIIA/genética , Factor de Transcripción TFIIB/química , Factor de Transcripción TFIIB/genéticaRESUMEN
Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a prerequisite for the biosynthesis of ribosomes in eukaryotes. Compared to Pols II and III, the mechanisms underlying promoter recognition, initiation complex formation and DNA melting by Pol I substantially diverge. Here, we report the high-resolution cryo-EM reconstruction of a Pol I early initiation intermediate assembled on a double-stranded promoter scaffold that prevents the establishment of downstream DNA contacts. Our analyses demonstrate how efficient promoter-backbone interaction is achieved by combined re-arrangements of flexible regions in the 'core factor' subunits Rrn7 and Rrn11. Furthermore, structure-function analysis illustrates how destabilization of the melted DNA region correlates with contraction of the polymerase cleft upon transcription activation, thereby combining promoter recruitment with DNA-melting. This suggests that molecular mechanisms and structural features of Pol I initiation have co-evolved to support the efficient melting, initial transcription and promoter clearance required for high-level rRNA synthesis.
Asunto(s)
Regiones Promotoras Genéticas , ARN Polimerasa I/metabolismo , Saccharomyces cerevisiae/genética , Iniciación de la Transcripción Genética , Secuencia de Aminoácidos , ADN/química , ADN/ultraestructura , Modelos Moleculares , Estructura Secundaria de Proteína , Subunidades de Proteína/metabolismo , ARN Polimerasa I/química , ARN Polimerasa I/ultraestructura , Factor de Transcripción TFIIB/metabolismoRESUMEN
In mammalian cells, widespread acceleration of cytoplasmic mRNA degradation is linked to impaired RNA polymerase II (Pol II) transcription. This mRNA decay-induced transcriptional repression occurs during infection with gammaherpesviruses including Kaposi's sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68), which encode an mRNA endonuclease that initiates widespread RNA decay. Here, we show that MHV68-induced mRNA decay leads to a genome-wide reduction of Pol II occupancy at mammalian promoters. This reduced Pol II occupancy is accompanied by down-regulation of multiple Pol II subunits and TFIIB in the nucleus of infected cells, as revealed by mass spectrometry-based global measurements of protein abundance. Viral genes, despite the fact that they require Pol II for transcription, escape transcriptional repression. Protection is not governed by viral promoter sequences; instead, location on the viral genome is both necessary and sufficient to escape the transcriptional repression effects of mRNA decay. We propose a model in which the ability to escape from transcriptional repression is linked to the localization of viral DNA within replication compartments, providing a means for these viruses to counteract decay-induced transcript loss.
Asunto(s)
Infecciones por Herpesviridae/metabolismo , Herpesvirus Humano 8/fisiología , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Estabilidad del ARN , Rhadinovirus/fisiología , Replicación Viral , Animales , Endonucleasas/genética , Endonucleasas/metabolismo , Genoma Viral , Infecciones por Herpesviridae/genética , Ratones , Células 3T3 NIH , ARN Polimerasa II/genética , Factor de Transcripción TFIIB/genética , Factor de Transcripción TFIIB/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Gene loops are formed by the interaction of initiation and termination factors occupying the distal ends of a gene during transcription. RNAPII is believed to affect gene looping indirectly owing to its essential role in transcription. The results presented here, however, demonstrate a direct role of RNAPII in gene looping through the Rpb4 subunit. 3C analysis revealed that gene looping is abolished in the rpb4Δ mutant. In contrast to the other looping-defective mutants, rpb4Δ cells do not exhibit a transcription termination defect. RPB4 overexpression, however, rescued the transcription termination and gene looping defect of sua7-1, a mutant of TFIIB. Furthermore, RPB4 overexpression rescued the ssu72-2 gene looping defect, while SSU72 overexpression restored the formation of gene loops in rpb4Δ cells. Interestingly, the interaction of TFIIB with Ssu72 is compromised in rpb4Δ cells. These results suggest that the TFIIB-Ssu72 interaction, which is critical for gene loop formation, is facilitated by Rpb4. We propose that Rpb4 is promoting the transfer of RNAPII from the terminator to the promoter for reinitiation of transcription through TFIIB-Ssu72 mediated gene looping.
Asunto(s)
ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factor de Transcripción TFIIB/metabolismo , Terminación de la Transcripción Genética , Genes Fúngicos , Modelos Genéticos , ARN Polimerasa II/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Iniciación de la Transcripción GenéticaRESUMEN
The TFIIB-related factor (BRF) family plays vital roles in RNA polymerase (Pol) III transcription initiation. However, little is known about the role of BRF in plants. Here, we report BRF1 and BRF2 are involved in Arabidopsis reproduction. In this study, we generated BRF1 and BRF2 double mutant plants. We found that no homozygous double mutants of brf1brf2 were obtained when brf1 and brf2 were crossed, although brf1 and brf2 mutants individually developed and reproduced normally. Further experiments revealed that heterozygous brf1/ + brf2/ + produced abnormal pollen and had no seeds in some placentas of siliques. Genetic data derived from reciprocal crosses showed that BRF2 plays a dominant role in Arabidopsis reproduction. Taken together, a double mutation of BRF1 and BRF2 results in a high degree of aborted macrogametes and microgametes and complete failure in zygote generation, ultimately leading to sterility.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Infertilidad Vegetal/genética , ARN Polimerasa III/genética , Factor de Transcripción TFIIB/genética , Factores de Transcripción/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cruzamientos Genéticos , Mutación , Polen/genética , Polen/metabolismo , Polen/ultraestructura , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Polimerasa III/metabolismo , Reproducción/genética , Factor de Transcripción TFIIB/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , CigotoRESUMEN
Transcription initiation can be reconstituted from highly purified general transcription factors (GTFs), RNA polymerase II (pol II), and promoter DNA. However, earlier biochemical reconstitution systems had a serious technical limitation, namely very poor initiation efficiency. Due to the poor efficiency of the reaction and trace amounts of proteins involved in the pre-initiation complex (PIC) assembly, detection of transcription and PIC formation was only possible by the synthesis of a radiolabeled transcript and by immunoblotting for PIC components on templates. Here we describe a transcription system that is capable of initiating transcription with >90% efficiency of template usage using homogeneous, active yeast components including TFIIA, TFIIB, TBP, TFIIE, TFIIF, TFIIH, Sub1, and pol II. The abundant specifically assembled PICs on promoter DNA can be separated from free general transcription factors (GTFs) and pol II by density gradient sedimentation, irrespective of the length of promoter DNA. The system is robust, and can be modified to accommodate many other transcription factors, and the resulting complexes can be analyzed by SDS-PAGE followed by Coomassie Blue staining. This technical advance now paves the way to conduct definitive biochemical and structural studies of the complete process of pol II initiation from the PIC, through promoter escape, and finally to productive elongation.
Asunto(s)
Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Factores de Transcripción/metabolismo , Iniciación de la Transcripción Genética , Complejos Multiproteicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Factor de Transcripción TFIIA/metabolismo , Factor de Transcripción TFIIB/metabolismo , Factores de Transcripción TFII/metabolismo , Levaduras/enzimología , Levaduras/genética , Levaduras/metabolismoRESUMEN
RNA polymerase II (Pol II) small nuclear RNA (snRNA) promoters and type 3 Pol III promoters have highly similar structures; both contain an interchangeable enhancer and "proximal sequence element" (PSE), which recruits the SNAP complex (SNAPc). The main distinguishing feature is the presence, in the type 3 promoters only, of a TATA box, which determines Pol III specificity. To understand the mechanism by which the absence or presence of a TATA box results in specific Pol recruitment, we examined how SNAPc and general transcription factors required for Pol II or Pol III transcription of SNAPc-dependent genes (i.e., TATA-box-binding protein [TBP], TFIIB, and TFIIA for Pol II transcription and TBP and BRF2 for Pol III transcription) assemble to ensure specific Pol recruitment. TFIIB and BRF2 could each, in a mutually exclusive fashion, be recruited to SNAPc. In contrast, TBP-TFIIB and TBP-BRF2 complexes were not recruited unless a TATA box was present, which allowed selective and efficient recruitment of the TBP-BRF2 complex. Thus, TBP both prevented BRF2 recruitment to Pol II promoters and enhanced BRF2 recruitment to Pol III promoters. On Pol II promoters, TBP recruitment was separate from TFIIB recruitment and enhanced by TFIIA. Our results provide a model for specific Pol recruitment at SNAPc-dependent promoters.
Asunto(s)
Regiones Promotoras Genéticas , ARN Polimerasa III/metabolismo , ARN Polimerasa II/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Células HEK293 , Humanos , Mutación , Unión Proteica , Dominios Proteicos , Transporte de Proteínas , TATA Box/genética , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIIB/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Viruses have evolved a plethora of mechanisms to target host antiviral responses. Here, we propose a yet uncharacterized mechanism of immune regulation by the orthomyxovirus Thogoto virus (THOV) ML protein through engaging general transcription factor TFIIB. ML generates a TFIIB depleted nuclear environment by re-localizing it into the cytoplasm. Although a broad effect on gene expression would be anticipated, ML expression, delivery of an ML-derived functional domain or experimental depletion of TFIIB only leads to altered expression of a limited number of genes. Our data indicate that TFIIB is critically important for the de novo recruitment of Pol II to promoter start sites and that TFIIB may not be required for regulated gene expression from paused promoters. Since many immune genes require de novo recruitment of Pol II, targeting of TFIIB by THOV represents a neat mechanism to affect immune responses while keeping other cellular transcriptional activities intact. Thus, interference with TFIIB activity may be a favourable site for therapeutic intervention to control undesirable inflammation.
Asunto(s)
Antivirales/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Gripe Humana/inmunología , Thogotovirus/inmunología , Factor de Transcripción TFIIB/metabolismo , Transcripción Genética/efectos de los fármacos , Proteínas Virales/metabolismo , Células HeLa , Humanos , Gripe Humana/tratamiento farmacológico , Gripe Humana/metabolismo , Regiones Promotoras Genéticas , Thogotovirus/efectos de los fármacos , Factor de Transcripción TFIIB/genéticaRESUMEN
Functional cross-talk between the promoter and terminator of a gene has long been noted. Promoters and terminators are juxtaposed to form gene loops in several organisms, and gene looping is thought to be involved in transcriptional regulation. The general transcription factor IIB (TFIIB) and the C-terminal domain phosphatase Ssu72, essential factors of the transcription preinitiation complex and the mRNA processing and polyadenylation complex, respectively, are important for gene loop formation. TFIIB and Ssu72 interact both genetically and physically, but the molecular basis of this interaction is not known. Here we present a crystal structure of the core domain of TFIIB in two new conformations that differ in the relative distance and orientation of the two cyclin-like domains. The observed extraordinary conformational plasticity may underlie the binding of TFIIB to multiple transcription factors and promoter DNAs that occurs in distinct stages of transcription, including initiation, reinitiation, and gene looping. We mapped the binding interface of the TFIIB-Ssu72 complex using a series of systematic, structure-guided in vitro binding and site-specific photocross-linking assays. Our results indicate that Ssu72 competes with acidic activators for TFIIB binding and that Ssu72 disrupts an intramolecular TFIIB complex known to impede transcription initiation. We also show that the TFIIB-binding site on Ssu72 overlaps with the binding site of symplekin, a component of the mRNA processing and polyadenylation complex. We propose a hand-off model in which Ssu72 mediates a conformational transition in TFIIB, accounting for the role of Ssu72 in transcription reinitiation, gene looping, and promoter-terminator cross-talk.
Asunto(s)
Proteínas Portadoras/química , Modelos Moleculares , Complejos Multiproteicos/química , Elementos de Respuesta , Factor de Transcripción TFIIB/química , Iniciación de la Transcripción Genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Fosfoproteínas Fosfatasas , Dominios Proteicos , Estructura Cuaternaria de Proteína , Factor de Transcripción TFIIB/genética , Factor de Transcripción TFIIB/metabolismoRESUMEN
While both archaeal and eukaryotic transcription initiation systems utilize TBP (TATA box-binding protein) and TFIIB (transcription factor IIB), eukaryotic systems include larger numbers of initiation factors. It remains uncertain how eukaryotic transcription initiation systems have evolved. Here, we investigate the evolutionary development of TBP and TFIIB, each of which has an intramolecular direct repeat, using two evolutionary indicators. Inter-repeat sequence dissimilarity (dDR, distance between direct repeats) indicates that the asymmetry of two repeats in TBP and TFIIB has gradually increased during evolution. Interspecies sequence diversity (PD, phylogenetic diversity) indicates that the resultant asymmetric structure, which is related to the ability to interact with multiple factors, diverged in archaeal TBP and archaeal/eukaryotic TFIIB during evolution. Our findings suggest that eukaryotic TBP initially acquired multiple Eukarya-specific interactors through asymmetric evolution of the two repeats. After the asymmetric TBP generated the complexity of the eukaryotic transcription initiation systems, its diversification halted and its asymmetric structure spread throughout eukaryotic species.