RESUMO
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.
Assuntos
Fosfoproteínas , Regiões Promotoras Genéticas , RNA Polimerase II , Proteína de Ligação a TATA-Box , Fator de Transcrição TFIIB , Fatores de Transcrição , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Humanos , Fator de Transcrição TFIIB/metabolismo , Fator de Transcrição TFIIB/genética , Proteína de Ligação a TATA-Box/metabolismo , Proteína de Ligação a TATA-Box/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Iniciação da Transcrição Genética , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Ligação Proteica , Fator de Transcrição TFIIA/metabolismo , Fator de Transcrição TFIIA/genética , Transcrição Gênica , Elongação da Transcrição Genética , RNA/metabolismo , RNA/genética , Fatores de Transcrição TFII/metabolismo , Fatores de Transcrição TFII/genéticaRESUMO
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.
Assuntos
Mapas de Interação de Proteínas , RNA Polimerase II , Fator de Transcrição TFIIB/genética , Fator de Transcrição TFIIB/metabolismo , RNA Polimerase II/genética , Proteômica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cromatina/genética , Cromatina/metabolismoRESUMO
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.
Assuntos
Neoplasias , Fatores Associados à Proteína de Ligação a TATA , Humanos , DNA Ribossômico/genética , Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Fator de Transcrição TFIIB/genética , Fator de Transcrição TFIIB/metabolismo , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Viroids, a fascinating group of plant pathogens, are subviral agents composed of single-stranded circular noncoding RNAs. It is well-known that nuclear-replicating viroids exploit host DNA-dependent RNA polymerase II (Pol II) activity for transcription from circular RNA genome to minus-strand intermediates, a classic example illustrating the intrinsic RNA-dependent RNA polymerase activity of Pol II. The mechanism for Pol II to accept single-stranded RNAs as templates remains poorly understood. Here, we reconstituted a robust in vitro transcription system and demonstrated that Pol II also accepts minus-strand viroid RNA template to generate plus-strand RNAs. Further, we purified the Pol II complex on RNA templates for nano-liquid chromatography-tandem mass spectrometry analysis and identified a remodeled Pol II missing Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9, contrasting to the canonical 12-subunit Pol II or the 10-subunit Pol II core on DNA templates. Interestingly, the absence of Rpb9, which is responsible for Pol II fidelity, explains the higher mutation rate of viroids in comparison to cellular transcripts. This remodeled Pol II is active for transcription with the aid of TFIIIA-7ZF and appears not to require other canonical general transcription factors (such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIS), suggesting a distinct mechanism/machinery for viroid RNA-templated transcription. Transcription elongation factors, such as FACT complex, PAF1 complex, and SPT6, were also absent in the reconstituted transcription complex. Further analyses of the critical zinc finger domains in TFIIIA-7ZF revealed the first three zinc finger domains pivotal for RNA template binding. Collectively, our data illustrated a distinct organization of Pol II complex on viroid RNA templates, providing new insights into viroid replication, the evolution of transcription machinery, as well as the mechanism of RNA-templated transcription.
Assuntos
Fatores Genéricos de Transcrição , Viroides , DNA/metabolismo , RNA/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Circular/genética , RNA Polimerase Dependente de RNA/genética , Fator de Transcrição TFIIA/genética , Fator de Transcrição TFIIA/metabolismo , Fator de Transcrição TFIIB/genética , Fator de Transcrição TFIID/genética , Fator de Transcrição TFIID/metabolismo , Fator de Transcrição TFIIIA/metabolismo , Fatores Genéricos de Transcrição/genética , Fatores Genéricos de Transcrição/metabolismo , Transcrição Gênica , Viroides/genética , Viroides/metabolismoRESUMO
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.
Assuntos
RNA Polimerase II , Fatores de Transcrição , Humanos , Fator de Transcrição TFIIB/genética , Fator de Transcrição TFIIB/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Fator de Transcrição TFIID/genética , Fator de Transcrição TFIID/metabolismo , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Transcrição Gênica , TATA Box/genética , RNA Polimerase III/genéticaRESUMO
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.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Fator de Transcrição TFIIB/genética , Fator de Transcrição TFIIB/metabolismo , Fator de Transcrição TFIID/genética , Fator de Transcrição TFIID/metabolismo , Transcrição GênicaRESUMO
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.
Assuntos
Eucariotos , RNA Polimerase II , Eucariotos/genética , Eucariotos/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/genética , Fator de Transcrição TFIIB/genética , Fator de Transcrição TFIIB/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
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.
Assuntos
DNA/metabolismo , Nucleossomos/metabolismo , Proteína de Ligação a TATA-Box/metabolismo , Fator de Transcrição TFIIA/metabolismo , Fator de Transcrição TFIIB/metabolismo , DNA/química , Modelos Moleculares , Nucleossomos/química , Ligação Proteica , Conformação Proteica , Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIIA/genética , Fator de Transcrição TFIIB/genéticaRESUMO
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.
Assuntos
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genoma Fúngico/genética , Complexos Multiproteicos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Coenzimas/metabolismo , Complexos Multiproteicos/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase I/metabolismo , RNA Polimerase II/metabolismo , RNA Polimerase III/metabolismo , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Fator de Transcrição TFIIB/genética , Fator de Transcrição TFIIB/metabolismo , Fator de Transcrição TFIID , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Sítios de Ligação , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Elementos de Resposta , Fator de Transcrição TFIIA/metabolismo , Fator de Transcrição TFIIB/metabolismo , Ativação Transcricional , Aminoácidos , Sequência de Bases , Linhagem Celular , Humanos , Modelos Moleculares , Mutação , Motivos de Nucleotídeos , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Fator de Transcrição TFIIA/química , Fator de Transcrição TFIIA/genética , Fator de Transcrição TFIIB/química , Fator de Transcrição TFIIB/genéticaRESUMO
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.
Assuntos
DNA/genética , RNA Polimerase II/genética , Fator de Transcrição TFIIB/genética , Transcrição Gênica/genética , Fluorescência , Humanos , Cinética , Regiões Promotoras Genéticas/genética , RNA/biossíntese , RNA/genética , Imagem Individual de Molécula/métodosRESUMO
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.
Assuntos
Infecções por Herpesviridae/metabolismo , Herpesvirus Humano 8/fisiologia , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Estabilidade de RNA , Rhadinovirus/fisiologia , Replicação Viral , Animais , Endonucleases/genética , Endonucleases/metabolismo , Genoma Viral , Infecções por Herpesviridae/genética , Camundongos , Células NIH 3T3 , RNA Polimerase II/genética , Fator de Transcrição TFIIB/genética , Fator de Transcrição TFIIB/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
In the absence of ligands, the nuclear receptor PPARß/δ recruits the NCOR and SMRT corepressors, which form complexes with HDAC3, to canonical target genes. Agonistic ligands cause dissociation of corepressors and enable enhanced transcription. Vice versa, synthetic inverse agonists augment corepressor recruitment and repression. Both basal repression of the target gene ANGPTL4 and reinforced repression elicited by inverse agonists are partially insensitive to HDAC inhibition. This raises the question how PPARß/δ represses transcription mechanistically. We show that the PPARß/δ inverse agonist PT-S264 impairs transcription initiation by decreasing recruitment of activating Mediator subunits, RNA polymerase II, and TFIIB, but not of TFIIA, to the ANGPTL4 promoter. Mass spectrometry identifies NCOR as the main PT-S264-dependent interactor of PPARß/δ. Reconstitution of knockout cells with PPARß/δ mutants deficient in basal repression results in diminished recruitment of NCOR, SMRT, and HDAC3 to PPAR target genes, while occupancy by RNA polymerase II is increased. PT-S264 restores binding of NCOR, SMRT, and HDAC3 to the mutants, resulting in reduced polymerase II occupancy. Our findings corroborate deacetylase-dependent and -independent repressive functions of HDAC3-containing complexes, which act in parallel to downregulate transcription.
Assuntos
Proteína 4 Semelhante a Angiopoietina/genética , Histona Desacetilases/genética , Complexos Multiproteicos/genética , PPAR beta/genética , Transcrição Gênica , Linhagem Celular , Humanos , Ligantes , Espectrometria de Massas , Correpressor 1 de Receptor Nuclear/genética , Correpressor 2 de Receptor Nuclear/genética , Regiões Promotoras Genéticas/genética , RNA Polimerase II/genética , Fator de Transcrição TFIIB/genética , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Infertilidade das Plantas/genética , RNA Polimerase III/genética , Fator de Transcrição TFIIB/genética , Fatores de Transcrição/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cruzamentos Genéticos , Mutação , Pólen/genética , Pólen/metabolismo , Pólen/ultraestrutura , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Polimerase III/metabolismo , Reprodução/genética , Fator de Transcrição TFIIB/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , ZigotoRESUMO
The present study reports the complete sequences of Aquaporin 1 (AQP1) gene and partial sequences of genes, Sodium/potassium-transporting ATPase subunit alpha-1 (Na/K-ATPase α1 subunit), Osmotic Stress Transcription Factor 1 (OSTF1), Transcription Factor II B (TFIIB), Heat Shock Cognate 71 (HSC71) and Heat Shock Protein 90 (HSP90) obtained from mRNA and genomic DNA of Etroplus suratensis. They are candidate genes involved in stress responses of fishes. AQP1 gene was 2163 bp long. Its mRNA sequence has 55 bp 5' UTR, 783 bp open reading frame (ORF), 119 bp 3' UTR, three intronic regions and 90% identity with AQP1 of Oreochromis niloticus. The partial Na/K-ATPase α1subunit gene obtained 5998 bp length with an ORF of 2213 bp and 12 intronic regions. The partial OSTF1, TF IIB, HSC71 and HSP90 mRNA sequences obtained were 1473 bp, 587 bp, 1708 bp and 151 bp in length respectively. All the genes showed a high sequence similarity with respective genes reported from fishes. Comparison of AQP1 and Na/K-ATPase α1 genomic DNA sequence of E. suratensis collected from different water system showed two type of AQP1 with one synonymous mutation in exon-1 and higher sequence difference in intronic regions (including addition, deletion, transition and transversion mutations) with few synonymous and non-synonymous mutations in the exons of Na/K-ATPase α1. The sequence information of these major candidate genes involved in stress responses will help in further studies on population genetics, adaptive variations and genetic improvement programs of this cichlid species having aquaculture, ornamental and evolutionary importance.
Assuntos
Ciclídeos/genética , Sequência de Aminoácidos , Animais , Aquaporina 1/química , Aquaporina 1/genética , Sequência de Bases/genética , Ciclídeos/fisiologia , Clonagem Molecular/métodos , Éxons , Regulação da Expressão Gênica/genética , Proteínas de Choque Térmico HSC70/genética , Proteínas de Choque Térmico HSP90/genética , Peptídeos e Proteínas de Sinalização Intracelular , Íntrons , Peptídeos/genética , ATPase Trocadora de Sódio-Potássio/genética , Fator de Transcrição TFIIB/genéticaRESUMO
Transcription initiation by archaeal RNA polymerase (RNAP) and eukaryotic RNAP II requires the general transcription factor (TF) B/ IIB. Structural analyses of eukaryotic transcription initiation complexes locate the B-reader domain of TFIIB in close proximity to the active site of RNAP II. Here, we present the first crosslinking mapping data that describe the dynamic transitions of an archaeal TFB to provide evidence for structural rearrangements within the transcription complex during transition from initiation to early elongation phase of transcription. Using a highly specific UV-inducible crosslinking system based on the unnatural amino acid para-benzoyl-phenylalanine allowed us to analyze contacts of the Pyrococcus furiosus TFB B-reader domain with site-specific radiolabeled DNA templates in preinitiation and initially transcribing complexes. Crosslink reactions at different initiation steps demonstrate interactions of TFB with DNA at registers +6 to +14, and reduced contacts at +15, with structural transitions of the B-reader domain detected at register +10. Our data suggest that the B-reader domain of TFB interacts with nascent RNA at register +6 and +8 and it is displaced from the transcribed-strand during the transition from +9 to +10, followed by the collapse of the transcription bubble and release of TFB from register +15 onwards.
Assuntos
Proteínas Arqueais/química , DNA/química , RNA Polimerase II/química , Fator de Transcrição TFIIB/química , Proteínas Arqueais/genética , DNA/genética , Domínios Proteicos , Pyrococcus furiosus/química , Pyrococcus furiosus/genética , RNA Polimerase II/genética , Fator de Transcrição TFIIB/genética , Transcrição GênicaRESUMO
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.
Assuntos
Antivirais/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Influenza Humana/imunologia , Thogotovirus/imunologia , Fator de Transcrição TFIIB/metabolismo , Transcrição Gênica/efeitos dos fármacos , Proteínas Virais/metabolismo , Células HeLa , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/metabolismo , Regiões Promotoras Genéticas , Thogotovirus/efeitos dos fármacos , Fator de Transcrição TFIIB/genéticaRESUMO
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.
Assuntos
Proteínas de Transporte/química , Modelos Moleculares , Complexos Multiproteicos/química , Elementos de Resposta , Fator de Transcrição TFIIB/química , Iniciação da Transcrição Genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fosfoproteínas Fosfatases , Domínios Proteicos , Estrutura Quaternária de Proteína , Fator de Transcrição TFIIB/genética , Fator de Transcrição TFIIB/metabolismoRESUMO
Eukaryotic mRNA transcription initiation is directed by the formation of the megadalton-sized preinitiation complex (PIC). After PIC formation, double-stranded DNA (dsDNA) is unwound to form a single-stranded DNA bubble, and the template strand is loaded into the polymerase active site. DNA opening is catalyzed by Ssl2 (XPB), the dsDNA translocase subunit of the basal transcription factor TFIIH. In yeast, transcription initiation proceeds through a scanning phase during which downstream DNA is searched for optimal start sites. Here, to test models for initial DNA opening and start-site scanning, we measure the DNA-bubble sizes generated by Saccharomyces cerevisiae PICs in real time using single-molecule magnetic tweezers. We show that ATP hydrolysis by Ssl2 opens a 6-base-pair (bp) bubble that grows to 13 bp in the presence of NTPs. These observations support a two-step model wherein ATP-dependent Ssl2 translocation leads to a 6-bp open complex that RNA polymerase II expands via NTP-dependent RNA transcription.
Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fator de Transcrição TFIIB/metabolismo , Fator de Transcrição TFIIH/metabolismo , Fatores de Transcrição TFII/metabolismo , Sítio de Iniciação de Transcrição/fisiologia , Iniciação da Transcrição Genética/fisiologia , Domínio Catalítico/genética , DNA Helicases/metabolismo , Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fator de Transcrição TFIIB/genética , Fator de Transcrição TFIIH/genética , Fatores de Transcrição TFII/genéticaRESUMO
Inteins are mobile genetic elements that are spliced out of proteins after translation. Some inteins contain a homing endonuclease (HEN) responsible for their propagation. Hedgehog/INTein (HINT) domains catalyzing protein splicing and their nested HEN domains are thought to be functionally independent because of the existence of functional mini-inteins without HEN domains. Despite the lack of obvious mutualism between HEN and HINT domains, HEN domains are persistently found at one specific site in inteins, indicating their potential functional role in protein splicing. Here we report crystal structures of inactive and active mini-inteins derived from inteins residing in the transcription factor IIB of Methanococcus jannaschii and Methanocaldococcus vulcanius, revealing a novel modified HINT fold that might provide new insights into the mutualism between the HEN and HINT domains. We propose an evolutionary model of inteins and a functional role of HEN domains in inteins.