RESUMO
During transcription, RNA polymerase II (RNAPII) faces numerous obstacles, including DNA damage, which can lead to stalling or arrest. One mechanism to contend with this situation is ubiquitylation and degradation of the largest RNAPII subunit, RPB1 - the 'last resort' pathway. This conserved, multi-step pathway was first identified in yeast, and the functional human orthologues of all but one protein, RNAPII Degradation Factor 1 (Def1), have been discovered. Here we show that following UV-irradiation, human Ubiquitin-associated protein 2 (UBAP2) or its paralogue UBAP2-like (UBAP2L) are involved in the ubiquitylation and degradation of RNAPII through the recruitment of Elongin-Cul5 ubiquitin ligase. Together, our data indicate that UBAP2 and UBAP2L are the human orthologues of yeast Def1, and so identify the key missing proteins in the human last resort pathway.
Assuntos
RNA Polimerase II , Proteínas de Saccharomyces cerevisiae , Humanos , Proteínas de Transporte/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Culina/metabolismo , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitinação , Raios UltravioletaRESUMO
Non-polyadenylated mRNAs of replication-dependent histones (RDHs) are synthesized by RNA polymerase II (Pol II) at histone locus bodies (HLBs). HLBs frequently associate with Cajal bodies (CBs), in which 3'-end processing factors for RDH genes are enriched; however, this association's role in transcription termination of RDH genes remains unclear. Here, we show that Pol II pauses immediately upstream of transcript end sites of RDH genes and Mediator plays a role in this Pol II pausing through CBs' association with HLBs. Disruption of the Mediator docking site for Little elongation complex (LEC)-Cap binding complex (CBC)-Negative elongation factor (NELF), components of CBs, interferes with CBs' association with HLBs and 3' Pol II pausing, resulting in increased aberrant unprocessed RDH gene transcripts. Our findings suggest Mediator's involvement in CBs' association with HLBs to facilitate 3' Pol II pausing and subsequent 3'-end processing of RDH genes by supplying 3'-end processing factors.
Assuntos
Corpos Enovelados , Histonas , Corpos Enovelados/metabolismo , Histonas/metabolismo , Corpos Nucleares , RNA Polimerase II/metabolismo , Transcrição GênicaRESUMO
The bZIP transcription factor ATF6α is a master regulator of endoplasmic reticulum (ER) stress response genes. In this report, we identify the multifunctional RNA polymerase II transcription factor Elongin as a cofactor for ATF6α-dependent transcription activation. Biochemical studies reveal that Elongin functions at least in part by facilitating ATF6α-dependent loading of Mediator at the promoters and enhancers of ER stress response genes. Depletion of Elongin from cells leads to impaired transcription of ER stress response genes and to defects in the recruitment of Mediator and its CDK8 kinase subunit. Taken together, these findings bring to light a role for Elongin as a loading factor for Mediator during the ER stress response.
Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Elonguina/metabolismo , Estresse do Retículo Endoplasmático , Regulação da Expressão Gênica , Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Fator 6 Ativador da Transcrição/genética , Animais , Elonguina/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Células HeLa , Humanos , Complexo Mediador/genética , Regiões Promotoras Genéticas , RNA Polimerase II/genética , Ratos , Transdução de Sinais , Ativação TranscricionalRESUMO
The SNF2 family ATPase Amplified in Liver Cancer 1 (ALC1) is the only chromatin remodeling enzyme with a poly(ADP-ribose) (PAR) binding macrodomain. ALC1 functions together with poly(ADP-ribose) polymerase PARP1 to remodel nucleosomes. Activation of ALC1 cryptic ATPase activity and the subsequent nucleosome remodeling requires binding of its macrodomain to PAR chains synthesized by PARP1 and NAD+ A key question is whether PARP1 has a role(s) in ALC1-dependent nucleosome remodeling beyond simply synthesizing the PAR chains needed to activate the ALC1 ATPase. Here, we identify PARP1 separation-of-function mutants that activate ALC1 ATPase but do not support nucleosome remodeling by ALC1. Investigation of these mutants has revealed multiple functions for PARP1 in ALC1-dependent nucleosome remodeling and provides insights into its multifaceted role in chromatin remodeling.
Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Nucleossomos/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , Reparo do DNA , HumanosRESUMO
The Elongin complex was originally identified as an RNA polymerase II (RNAPII) elongation factor and subsequently as the substrate recognition component of a Cullin-RING E3 ubiquitin ligase. More recent evidence indicates that the Elongin ubiquitin ligase assembles with the Cockayne syndrome B helicase (CSB) in response to DNA damage and can target stalled polymerases for ubiquitylation and removal from the genome. In this report, we present evidence that the CSB-Elongin ubiquitin ligase pathway has roles beyond the DNA damage response in the activation of RNAPII-mediated transcription. We observed that assembly of the CSB-Elongin ubiquitin ligase is induced not just by DNA damage, but also by a variety of signals that activate RNAPII-mediated transcription, including endoplasmic reticulum (ER) stress, amino acid starvation, retinoic acid, glucocorticoids, and doxycycline treatment of cells carrying several copies of a doxycycline-inducible reporter. Using glucocorticoid receptor (GR)-regulated genes as a model, we showed that glucocorticoid-induced transcription is accompanied by rapid recruitment of CSB and the Elongin ubiquitin ligase to target genes in a step that depends upon the presence of transcribing RNAPII on those genes. Consistent with the idea that the CSB-Elongin pathway plays a direct role in GR-regulated transcription, mouse cells lacking the Elongin subunit Elongin A exhibit delays in both RNAPII accumulation on and dismissal from target genes following glucocorticoid addition and withdrawal, respectively. Taken together, our findings bring to light a new role for the CSB-Elongin pathway in RNAPII-mediated transcription.
Assuntos
DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Elonguina/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Polimerase II/genética , Ubiquitina-Proteína Ligases/genética , Animais , Síndrome de Cockayne/enzimologia , Síndrome de Cockayne/genética , DNA Helicases/química , DNA Helicases/ultraestrutura , Reparo do DNA/genética , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/ultraestrutura , Elonguina/química , Elonguina/ultraestrutura , Humanos , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Proteínas de Ligação a Poli-ADP-Ribose/química , Proteínas de Ligação a Poli-ADP-Ribose/ultraestrutura , RNA Polimerase II/química , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/genética , Ubiquitina/química , Ubiquitina/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/ultraestrutura , Ubiquitinação/genéticaRESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disease caused by accumulations of Aß peptides. Production and fibrillation of Aß are downregulated by BRI2 and BRI3, which are physiological inhibitors of amyloid precursor protein (APP) processing and Aß oligomerization. Here, we identify nuclear receptor binding protein 1 (NRBP1) as a substrate receptor of a Cullin-RING ubiquitin ligase (CRL) that targets BRI2 and BRI3 for degradation. Moreover, we demonstrate that (1) dimerized NRBP1 assembles into a functional Cul2- and Cul4A-containing heterodimeric CRL through its BC-box and an overlapping cryptic H-box, (2) both Cul2 and Cul4A contribute to NRBP1 CRL function, and (3) formation of the NRBP1 heterodimeric CRL is strongly enhanced by chaperone-like function of TSC22D3 and TSC22D4. NRBP1 knockdown in neuronal cells results in an increase in the abundance of BRI2 and BRI3 and significantly reduces Aß production. Thus, disrupting interactions between NRBP1 and its substrates BRI2 and BRI3 may provide a useful therapeutic strategy for AD.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeos beta-Amiloides/biossíntese , Proteínas Culina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteólise , Receptores Citoplasmáticos e Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/metabolismo , Animais , Feminino , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos Endogâmicos ICR , Ligação Proteica , Multimerização Proteica , Receptores Citoplasmáticos e Nucleares/química , Especificidade por Substrato , Fatores de Transcrição/metabolismo , Ubiquitinação , Proteínas de Transporte Vesicular/químicaRESUMO
In response to transcription-blocking DNA damage, cells orchestrate a multi-pronged reaction, involving transcription-coupled DNA repair, degradation of RNA polymerase II (RNAPII), and genome-wide transcription shutdown. Here, we provide insight into how these responses are connected by the finding that ubiquitylation of RNAPII itself, at a single lysine (RPB1 K1268), is the focal point for DNA-damage-response coordination. K1268 ubiquitylation affects DNA repair and signals RNAPII degradation, essential for surviving genotoxic insult. RNAPII degradation results in a shutdown of transcriptional initiation, in the absence of which cells display dramatic transcriptome alterations. Additionally, regulation of RNAPII stability is central to transcription recovery-persistent RNAPII depletion underlies the failure of this process in Cockayne syndrome B cells. These data expose regulation of global RNAPII levels as integral to the cellular DNA-damage response and open the intriguing possibility that RNAPII pool size generally affects cell-specific transcription programs in genome instability disorders and even normal cells.
Assuntos
Dano ao DNA , RNA Polimerase II/metabolismo , Reparo do DNA , Células HEK293 , Humanos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Ubiquitinação , Raios UltravioletaRESUMO
Mediator is a coregulatory complex that regulates transcription of Pol II-dependent genes. Previously, we showed that human Mediator subunit MED26 plays a role in the recruitment of Super Elongation Complex (SEC) or Little Elongation Complex (LEC) to regulate the expression of certain genes. MED26 plays a role in recruiting SEC to protein-coding genes including c-myc and LEC to small nuclear RNA (snRNA) genes. However, how MED26 engages SEC or LEC to regulate distinct genes is unclear. Here, we provide evidence that MED26 recruits LEC to modulate transcription termination of non-polyadenylated transcripts including snRNAs and mRNAs encoding replication-dependent histone (RDH) at Cajal bodies. Our findings indicate that LEC recruited by MED26 promotes efficient transcription termination by Pol II through interaction with CBC-ARS2 and NELF/DSIF, and promotes 3' end processing by enhancing recruitment of Integrator or Heat Labile Factor to snRNA or RDH genes, respectively.
Assuntos
Regulação da Expressão Gênica/genética , Complexo Mediador/genética , RNA Nuclear Pequeno/genética , Terminação da Transcrição Genética/fisiologia , Fatores de Elongação da Transcrição/genética , Linhagem Celular Tumoral , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Proteínas de Ligação ao Cap de RNA/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/metabolismoRESUMO
The discovery of the three eukaryotic nuclear RNA polymerases paved the way for serious biochemical investigations of eukaryotic transcription and the identification of eukaryotic transcription factors. Here we describe this adventure from our vantage point, with a focus on the hunt for factors that regulate elongation by RNA polymerase II.
Assuntos
Bioquímica/história , Eucariotos/enzimologia , Biologia Molecular/história , RNA Polimerase II/metabolismo , Transcrição Gênica , Fatores de Elongação da Transcrição/isolamento & purificação , Fatores de Elongação da Transcrição/metabolismo , História do Século XX , História do Século XXIRESUMO
FACT (facilitates chromatin transcription) is an evolutionarily conserved histone chaperone that was initially identified as an activity capable of promoting RNA polymerase II (Pol II) transcription through nucleosomes in vitro. In this report, we describe a global analysis of FACT function in Pol II transcription in Drosophila. We present evidence that loss of FACT has a dramatic impact on Pol II elongation-coupled processes including histone H3 lysine 4 (H3K4) and H3K36 methylation, consistent with a role for FACT in coordinating histone modification and chromatin architecture during Pol II transcription. Importantly, we identify a role for FACT in the maintenance of promoter-proximal Pol II pausing, a key step in transcription activation in higher eukaryotes. These findings bring to light a broader role for FACT in the regulation of Pol II transcription.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Animais , Proteínas de Transporte/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Histonas/genética , RNA Polimerase II/genéticaRESUMO
Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA polymerase II and co-transcriptional capping and splicing of the precursor RNA to form the mature mRNA. During mRNA synthesis, the RNA polymerase II elongation complex is a target for regulation by a large collection of transcription factors that control its catalytic activity, as well as the capping, splicing, and 3'-processing enzymes that create the mature mRNA. Because of the inherent complexity of mRNA synthesis, simpler experimental systems enabling isolation and investigation of its various co-transcriptional stages have great utility. In this article, we describe one such simple experimental system suitable for investigating co-transcriptional RNA capping. This system relies on defined RNA polymerase II elongation complexes assembled from purified polymerase and artificial transcription bubbles. When immobilized via biotinylated DNA, these RNA polymerase II elongation complexes provide an easily manipulable tool for dissecting co-transcriptional RNA capping and mechanisms by which the elongation complex recruits and regulates capping enzyme during co-transcriptional RNA capping. We anticipate this system could be adapted for studying recruitment and/or assembly of proteins or protein complexes with roles in other stages of mRNA maturation coupled to the RNA polymerase II elongation complex.
Assuntos
RNA Polimerase II/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Animais , Humanos , Capuzes de RNA/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/genética , Ratos , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Elongin A binds to Elongins B and C to form the RNA polymerase II transcription elongation factor Elongin. It also functions as the substrate recognition subunit of a ubiquitin ligase that is formed by binding of Elongin to Cullin protein CUL5 and RING finger protein RBX2 and that targets RNA polymerase II for ubiquitination. In this article, we describe use of acceptor photobleaching fluorescence resonance energy transfer (AP-FRET) and laser microirradiation-based assays to study regulated assembly of the Elongin ubiquitin ligase and its recruitment to regions of localized DNA damage.
Assuntos
Dano ao DNA , Elonguina/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Culina/metabolismo , DNA/metabolismo , DNA/efeitos da radiação , Eucariotos/enzimologia , Eucariotos/metabolismo , LasersRESUMO
In a recent publication in Science, Kujirai et al. (2018) use single-particle cryo-EM to resolve several Pol II-nucleosome interactions, shedding new light on transcription elongation in a native chromatin environment.
Assuntos
Nucleossomos , RNA Polimerase II , Cromatina , Microscopia Crioeletrônica , Transcrição GênicaRESUMO
ELL family transcription factors activate the overall rate of RNA polymerase II (Pol II) transcription elongation by binding directly to Pol II and suppressing its tendency to pause. In metazoa, ELL regulates Pol II transcription elongation as part of a large multisubunit complex referred to as the Super Elongation Complex (SEC), which includes P-TEFb and EAF, AF9 or ENL, and an AFF family protein. Although orthologs of ELL and EAF have been identified in lower eukaryotes including Schizosaccharomyces pombe, it has been unclear whether SEC-like complexes function in lower eukaryotes. In this report, we describe isolation from S. pombe of an ELL-containing complex with features of a rudimentary SEC. This complex includes S. pombe Ell1, Eaf1, and a previously uncharacterized protein we designate Ell1 binding protein 1 (Ebp1), which is distantly related to metazoan AFF family members. Like the metazoan SEC, this S. pombe ELL complex appears to function broadly in Pol II transcription. Interestingly, it appears to have a particularly important role in regulating genes involved in cell separation.
Assuntos
RNA Polimerase II/genética , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/genética , Fatores de Elongação da Transcrição/genética , Fator B de Elongação Transcricional Positiva/química , Fator B de Elongação Transcricional Positiva/genética , RNA Polimerase II/química , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Fatores de Transcrição/química , Transcrição Gênica , Fatores de Elongação da Transcrição/químicaRESUMO
Co-transcriptional capping of RNA polymerase II (Pol II) transcripts by capping enzyme proceeds orders of magnitude more efficiently than capping of free RNA. Previous studies brought to light a role for the phosphorylated Pol II carboxyl-terminal domain (CTD) in activation of co-transcriptional capping; however, CTD phosphorylation alone could not account for the observed magnitude of activation. Here, we exploit a defined Pol II transcription system that supports both CTD phosphorylation and robust activation of capping to dissect the mechanism of co-transcriptional capping. Taken together, our findings identify a CTD-independent, but Pol II-mediated, mechanism that functions in parallel with CTD-dependent processes to ensure optimal capping, and they support a "tethering" model for the mechanism of activation.
Assuntos
RNA Polimerase II/química , RNA Polimerase II/metabolismo , Transcrição Gênica , Sequência de Bases , Quinases Ciclina-Dependentes/metabolismo , Humanos , Modelos Biológicos , Fosforilação , Domínios Proteicos , Capuzes de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade da Espécie , Relação Estrutura-Atividade , Fator de Transcrição TFIIH/metabolismo , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Although cell metabolism has been established as a major regulator of eukaryotic gene expression, the mechanisms underlying this regulation are still being uncovered. Recent years have seen great advances in our understanding of biochemical mechanisms of metabolic regulation of transcription and chromatin. Prime examples include insights into how nutrients and cellular energy status regulate synthesis of ribosomal RNAs by RNA polymerases I and III during ribosome biogenesis and how a variety of enzymes that catalyze modifications of histones in chromatin are regulated by the levels of certain metabolites. This volume of the Annual Review of Biochemistry includes a set of reviews describing these and other advances in understanding aspects of the metabolic regulation of RNA polymerases I and III transcription and chromatin.
Assuntos
Cromatina/metabolismo , Transcrição Gênica , Animais , RNA Polimerases Dirigidas por DNA/metabolismo , HumanosRESUMO
The human males absent on the first (MOF)-containing histone acetyltransferase nonspecific lethal (NSL) complex comprises nine subunits including the O-linked N-acetylglucosamine (O-GlcNAc) transferase, isoform 1 (OGT1). However, whether the O-GlcNAc transferase activity of OGT1 controls histone acetyltransferase activity of the NSL complex and whether OGT1 physically interacts with the other NSL complex subunits remain unclear. Here, we demonstrate that OGT1 regulates the activity of the NSL complex by mainly acetylating histone H4 Lys-16, Lys-5, and Lys-8 via O-GlcNAcylation and stabilization of the NSL complex subunit NSL3. Knocking down or overexpressing OGT1 in human cells remarkably affected the global acetylation of histone H4 residues Lys-16, Lys-5, and Lys-8. Because OGT1 is a subunit of the NSL complex, we also investigated the function of OGT1 in this complex. Co-transfection/co-immunoprecipitation experiments combined with in vitro O-GlcNAc transferase assays confirmed that OGT1 specifically binds to and O-GlcNAcylates NSL3. In addition, wheat germ agglutinin affinity purification verified the occurrence of O-GlcNAc modification on NSL3 in cells. Moreover, O-GlcNAcylation of NSL3 by wild-type OGT1 (OGT1-WT) stabilized NSL3. This stabilization was lost after co-transfection of NSL3 with an OGT1 mutant, OGT1C964A, that lacks O-GlcNAc transferase activity. Furthermore, stabilization of NSL3 by OGT1-WT significantly increased the global acetylation levels of H4 Lys-5, Lys-8, and Lys-16 in cells. These results suggest that OGT1 regulates the activity of the NSL complex by stabilizing NSL3.
Assuntos
Histona Acetiltransferases/metabolismo , Histonas/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Acetilação , Substituição de Aminoácidos , Animais , Células HEK293 , Células HeLa , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/química , N-Acetilglucosaminiltransferases/genética , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Proteínas Nucleares/genética , Mutação Puntual , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Especificidade por SubstratoRESUMO
Elongin A performs dual functions as the transcriptionally active subunit of RNA polymerase II (Pol II) elongation factor Elongin and as the substrate recognition subunit of a Cullin-RING E3 ubiquitin ligase that ubiquitylates Pol II in response to DNA damage. Assembly of the Elongin A ubiquitin ligase and its recruitment to sites of DNA damage is a tightly regulated process induced by DNA-damaging agents and α-amanitin, a drug that induces Pol II stalling. In this study, we demonstrate (i) that Elongin A and the ubiquitin ligase subunit CUL5 associate in cells with the Cockayne syndrome B (CSB) protein and (ii) that this interaction is also induced by DNA-damaging agents and α-amanitin. In addition, we present evidence that the CSB protein promotes stable recruitment of the Elongin A ubiquitin ligase to sites of DNA damage. Our findings are consistent with the model that the Elongin A ubiquitin ligase and the CSB protein function together in a common pathway in response to Pol II stalling and DNA damage.
Assuntos
Dano ao DNA , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Alfa-Amanitina/metabolismo , Linhagem Celular , Proteínas Culina/metabolismo , Reparo do DNA , Elonguina , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Mutação , Plasmídeos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Fatores de Transcrição/genéticaRESUMO
The conserved Mediator co-activator complex has an essential role in the regulation of RNA polymerase II transcription in all eukaryotes. Understanding the structure and interactions of Mediator is crucial for determining how the complex influences transcription initiation and conveys regulatory information to the basal transcription machinery. Here we present a 4.4 Å resolution cryo-electron microscopy map of Schizosaccharomyces pombe Mediator in which conserved Mediator subunits are individually resolved. The essential Med14 subunit works as a central backbone that connects the Mediator head, middle and tail modules. Comparison with a 7.8 Å resolution cryo-electron microscopy map of a Mediator-RNA polymerase II holoenzyme reveals that changes in the structure of Med14 facilitate a large-scale Mediator rearrangement that is essential for holoenzyme formation. Our study suggests that access to different conformations and crosstalk between structural elements are essential for the Mediator regulation mechanism, and could explain the capacity of the complex to integrate multiple regulatory signals.