RESUMO
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.
Assuntos
Microscopia Crioeletrônica , Complexo Mediador/química , Complexo Mediador/ultraestrutura , RNA Polimerase II/química , RNA Polimerase II/ultraestrutura , Regulação Alostérica , Sítios de Ligação , Domínio Catalítico , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , DNA Complementar/genética , Humanos , Complexo Mediador/metabolismo , Modelos Moleculares , Fosforilação , Ligação Proteica , RNA Polimerase II/metabolismo , Fator de Transcrição TFIIB/química , Fator de Transcrição TFIIB/metabolismo , Fatores de Transcrição TFII/química , Fatores de Transcrição TFII/metabolismo , Iniciação da Transcrição Genética , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
The Mediator complex plays an essential and multi-faceted role in regulation of RNA polymerase II transcription in all eukaryotes. Structural analysis of yeast Mediator has provided an understanding of the conserved core of the complex and its interaction with RNA polymerase II but failed to reveal the structure of the Tail module that contains most subunits targeted by activators and repressors. Here we present a molecular model of mammalian (Mus musculus) Mediator, derived from a 4.0 Å resolution cryo-EM map of the complex. The mammalian Mediator structure reveals that the previously unresolved Tail module, which includes a number of metazoan specific subunits, interacts extensively with core Mediator and has the potential to influence its conformation and interactions.
Assuntos
Sequência Conservada , Mamíferos/metabolismo , Complexo Mediador/química , Complexo Mediador/metabolismo , Animais , Linhagem Celular Tumoral , Doença/genética , Complexo Mediador/ultraestrutura , Camundongos , Modelos Moleculares , Mutação/genética , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Dysfunctions of nuclear processes including transcription and DNA repair lead to severe human diseases. Gaining an understanding of how these processes operate in the crowded context of chromatin can be particularly challenging. Mediator is a large multiprotein complex conserved in eukaryotes with a key coactivator role in the regulation of RNA polymerase (Pol) II transcription. Despite intensive studies, the molecular mechanisms underlying Mediator function remain to be fully understood. Novel findings have provided insights into the relationship between Mediator and chromatin architecture, revealed its role in connecting transcription with DNA repair and proposed an emerging mechanism of phase separation involving Mediator condensates. Recent developments in the field suggest multiple functions of Mediator going beyond transcriptional processes per se that would explain its involvement in various human pathologies.
Assuntos
Cromatina/genética , Complexo Mediador/genética , RNA Polimerase II/genética , Transcrição Gênica/genética , Cromatina/ultraestrutura , Reparo do DNA/genética , Humanos , Complexo Mediador/ultraestrutura , RNA Polimerase II/ultraestruturaRESUMO
For the initiation of transcription, RNA polymerase II (Pol II) assembles with general transcription factors on promoter DNA to form the pre-initiation complex (PIC). Here we report cryo-electron microscopy structures of the Saccharomyces cerevisiae PIC and PIC-core Mediator complex at nominal resolutions of 4.7 Å and 5.8 Å, respectively. The structures reveal transcription factor IIH (TFIIH), and suggest how the core and kinase TFIIH modules function in the opening of promoter DNA and the phosphorylation of Pol II, respectively. The TFIIH core subunit Ssl2 (a homologue of human XPB) is positioned on downstream DNA by the 'E-bridge' helix in TFIIE, consistent with TFIIE-stimulated DNA opening. The TFIIH kinase module subunit Tfb3 (MAT1 in human) anchors the kinase Kin28 (CDK7), which is mobile in the PIC but preferentially located between the Mediator hook and shoulder in the PIC-core Mediator complex. Open spaces between the Mediator head and middle modules may allow access of the kinase to its substrate, the C-terminal domain of Pol II.
Assuntos
Microscopia Crioeletrônica , Complexo Mediador/química , Complexo Mediador/ultraestrutura , Saccharomyces cerevisiae , Fatores de Transcrição TFII/química , Fatores de Transcrição TFII/ultraestrutura , Iniciação da Transcrição Genética , DNA/química , DNA/genética , DNA/metabolismo , Complexo Mediador/metabolismo , Modelos Moleculares , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Fatores de Transcrição TFII/metabolismoRESUMO
Mediator is a multiprotein co-activator that binds the transcription pre-initiation complex (PIC) and regulates RNA polymerase (Pol) II. The Mediator head and middle modules form the essential core Mediator (cMed), whereas the tail and kinase modules play regulatory roles. The architecture of Mediator and its position on the PIC are known, but atomic details are limited to Mediator subcomplexes. Here we report the crystal structure of the 15-subunit cMed from Schizosaccharomyces pombe at 3.4 Å resolution. The structure shows an unaltered head module, and reveals the intricate middle module, which we show is globally required for transcription. Sites of known Mediator mutations cluster at the interface between the head and middle modules, and in terminal regions of the head subunits Med6 (ref. 16) and Med17 (ref. 17) that tether the middle module. The structure led to a model for Saccharomyces cerevisiae cMed that could be combined with the 3.6 Å cryo-electron microscopy structure of the core PIC (cPIC). The resulting atomic model of the cPIC-cMed complex informs on interactions of the submodules forming the middle module, called beam, knob, plank, connector, and hook. The hook is flexibly linked to Mediator by a conserved hinge and contacts the transcription initiation factor IIH (TFIIH) kinase that phosphorylates the carboxy (C)-terminal domain (CTD) of Pol II and was recently positioned on the PIC. The hook also contains residues that crosslink to the CTD and reside in a previously described cradle. These results provide a framework for understanding Mediator function, including its role in stimulating CTD phosphorylation by TFIIH.
Assuntos
Microscopia Crioeletrônica , Complexo Mediador/química , RNA Polimerase II/química , Schizosaccharomyces/química , Fatores de Transcrição TFII/ultraestrutura , Iniciação da Transcrição Genética , Cristalografia por Raios X , Complexo Mediador/genética , Complexo Mediador/metabolismo , Complexo Mediador/ultraestrutura , Modelos Moleculares , Mutação , Fosforilação , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Schizosaccharomyces/ultraestrutura , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/ultraestrutura , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição TFII/química , Fatores de Transcrição TFII/metabolismoRESUMO
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.
Assuntos
Complexo Mediador/química , Complexo Mediador/metabolismo , RNA Polimerase II/química , RNA Polimerase II/ultraestrutura , Sítios de Ligação , Microscopia Crioeletrônica , Holoenzimas/química , Holoenzimas/metabolismo , Holoenzimas/ultraestrutura , Complexo Mediador/ultraestrutura , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA Polimerase II/metabolismo , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/ultraestrutura , Relação Estrutura-AtividadeRESUMO
The conserved co-activator complex Mediator enables regulated transcription initiation by RNA polymerase (Pol) II. Here we reconstitute an active 15-subunit core Mediator (cMed) comprising all essential Mediator subunits from Saccharomyces cerevisiae. The cryo-electron microscopic structure of cMed bound to a core initiation complex was determined at 9.7 Å resolution. cMed binds Pol II around the Rpb4-Rpb7 stalk near the carboxy-terminal domain (CTD). The Mediator head module binds the Pol II dock and the TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module extends to the Pol II foot with a 'plank' that may influence polymerase conformation. The Mediator subunit Med14 forms a 'beam' between the head and middle modules and connects to the tail module that is predicted to bind transcription activators located on upstream DNA. The Mediator 'arm' and 'hook' domains contribute to a 'cradle' that may position the CTD and TFIIH kinase to stimulate Pol II phosphorylation.
Assuntos
Microscopia Crioeletrônica , Complexo Mediador/química , Complexo Mediador/ultraestrutura , RNA Polimerase II/química , RNA Polimerase II/ultraestrutura , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestrutura , Regulação Alostérica , Sítios de Ligação , DNA/química , DNA/metabolismo , Ativação Enzimática , Complexo Mediador/metabolismo , Modelos Moleculares , Fosforilação , Estabilidade Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Fator de Transcrição TFIIB/química , Fator de Transcrição TFIIB/metabolismo , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo , Iniciação da Transcrição GenéticaRESUMO
The multisubunit Mediator, comprising â¼30 distinct proteins, plays an essential role in gene expression regulation by acting as a bridge between DNA-binding transcription factors and the RNA polymerase II (RNAPII) transcription machinery. Efforts to uncover the Mediator mechanism have been hindered by a poor understanding of its structure, subunit organization, and conformational rearrangements. By overcoming biochemical and image analysis hurdles, we obtained accurate EM structures of yeast and human Mediators. Subunit localization experiments, docking of partial X-ray structures, and biochemical analyses resulted in comprehensive mapping of yeast Mediator subunits and a complete reinterpretation of our previous Mediator organization model. Large-scale Mediator rearrangements depend on changes at the interfaces between previously described Mediator modules, which appear to be facilitated by factors conducive to transcription initiation. Conservation across eukaryotes of Mediator structure, subunit organization, and RNA polymerase II interaction suggest conservation of fundamental aspects of the Mediator mechanism.
Assuntos
Complexo Mediador/química , Complexo Mediador/ultraestrutura , Microscopia Crioeletrônica , Células HeLa , Humanos , Complexo Mediador/metabolismo , Modelos Moleculares , Mapeamento de Interação de Proteínas , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Mediator is a large and structurally dynamic protein complex that is globally required for eukaryotic transcription by RNA polymerase II. In a recent paper published in Cell Research, Wang et al. report for the first time the location of distinct subunits and redefine domains in the S. cerevisiae Mediator complex.
Assuntos
Complexo Mediador/ultraestrutura , Subunidades Proteicas/metabolismoRESUMO
The Mediator complex plays an essential role in the regulation of eukaryotic transcription. The Saccharomyces cerevisiae core Mediator comprises 21 subunits, which are organized into Head, Middle and Tail modules. Previously, the Head module was assigned to a distinct dense domain at the base, and the Middle and Tail modules were identified to form a tight structure above the Head module, which apparently contradicted findings from many biochemical and functional studies. Here, we compared the structures of the core Mediator and its subcomplexes, especially the first 3D structure of the Head + Middle modules, which permitted an unambiguous assignment of the three modules. Furthermore, nanogold labeling pinpointing four Mediator subunits from different modules conclusively validated the modular assignment, in which the Head and Middle modules fold back on one another and form the upper portion of the core Mediator, while the Tail module forms a distinct dense domain at the base. The new modular model of the core Mediator has reconciled the previous inconsistencies between the structurally and functionally defined Mediator modules. Collectively, these analyses completely redefine the modular organization of the core Mediator, which allow us to integrate the structural and functional information into a coherent mechanism for the Mediator's modularity and regulation in transcription initiation.
Assuntos
Complexo Mediador/ultraestrutura , Subunidades Proteicas/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Complexo Mediador/metabolismo , Modelos Moleculares , Conformação Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/ultraestruturaRESUMO
Mediator, a large (21 polypeptides, MW â¼1 MDa) complex conserved throughout eukaryotes, plays an essential role in control of gene expression by conveying regulatory signals that influence the activity of the preinitiation complex. However, the precise mode of interaction between Mediator and RNA polymerase II (RNAPII), and the mechanism of regulation by Mediator remain elusive. We used cryo-electron microscopy and reconstituted in vitro transcription assays to characterize a transcriptionally-active complex including the Mediator Head module and components of a minimum preinitiation complex (RNAPII, TFIIF, TFIIB, TBP, and promoter DNA). Our results reveal how the Head interacts with RNAPII, affecting its conformation and function.
Assuntos
Complexo Mediador/química , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Complexo Mediador/metabolismo , Complexo Mediador/ultraestrutura , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição TFII/química , Fatores de Transcrição TFII/metabolismoRESUMO
The human Mediator complex controls RNA polymerase II (pol II) function in ways that remain incompletely understood. Activator-Mediator binding alters Mediator structure, and these activator-induced structural shifts appear to play key roles in regulating transcription. A recent cryo-electron microscopy (EM) analysis revealed that pol II adopted a stable orientation within a Mediator-pol II-TFIIF assembly in which Mediator was bound to the activation domain of viral protein 16 (VP16). Whereas TFIIF was shown to be important for orienting pol II within this assembly, the potential role of the activator was not assessed. To determine how activator binding might affect pol II orientation, we isolated human Mediator-pol II-TFIIF complexes in which Mediator was not bound to an activator. Cryo-EM analysis of this assembly, coupled with pol II crystal structure docking, revealed that pol II binds Mediator at the same general location; however, in contrast to VP16-bound Mediator, pol II does not appear to stably orient in the absence of an activator. Variability in pol II orientation might be important mechanistically, perhaps to enable sense and antisense transcription at human promoters. Because Mediator interacts extensively with pol II, these results suggest that Mediator structural shifts induced by activator binding help stably orient pol II prior to transcription initiation.
Assuntos
Complexo Mediador/química , Complexo Mediador/metabolismo , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Fatores de Transcrição TFII/química , Fatores de Transcrição TFII/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Complexo Mediador/isolamento & purificação , Complexo Mediador/ultraestrutura , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Multimerização Proteica , RNA Polimerase II/isolamento & purificação , RNA Polimerase II/ultraestrutura , Fatores de Transcrição TFII/isolamento & purificação , Fatores de Transcrição TFII/ultraestruturaRESUMO
It is not well understood how the human Mediator complex, transcription factor IIH and RNA polymerase II (Pol II) work together with activators to initiate transcription. Activator binding alters Mediator structure, yet the functional consequences of such structural shifts remain unknown. The p53 C terminus and its activation domain interact with different Mediator subunits, and we find that each interaction differentially affects Mediator structure; strikingly, distinct p53-Mediator structures differentially affect Pol II activity. Only the p53 activation domain induces the formation of a large pocket domain at the Mediator-Pol II interaction site, and this correlates with activation of stalled Pol II to a productively elongating state. Moreover, we define a Mediator requirement for TFIIH-dependent Pol II C-terminal domain phosphorylation and identify substantial differences in Pol II C-terminal domain processing that correspond to distinct p53-Mediator structural states. Our results define a fundamental mechanism by which p53 activates transcription and suggest that Mediator structural shifts trigger activation of stalled Pol II complexes.
Assuntos
Complexo Mediador/química , Complexo Mediador/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Microscopia Crioeletrônica , Genes p53 , Células HeLa , Humanos , Técnicas In Vitro , Complexo Mediador/genética , Complexo Mediador/ultraestrutura , Modelos Biológicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , RNA Polimerase II/química , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/ultraestruturaRESUMO
We used single-particle electron microscopy to characterize the structure and subunit organization of the Mediator Head module that controls Mediator-RNA polymerase II (RNAPII) and Mediator-promoter interactions. The Head module adopts several conformations differing in the position of a movable jaw formed by the Med18-Med20 subcomplex. We also characterized, by structural, biochemical and genetic means, the interactions of the Head module with TATA-binding protein (TBP) and RNAPII subunits Rpb4 and Rpb7. TBP binds near the Med18-Med20 attachment point and stabilizes an open conformation of the Head module. Rpb4 and Rpb7 bind between the Head jaws, establishing contacts essential for yeast-cell viability. These results, and consideration of the structure of the Mediator-RNAPII holoenzyme, shed light on the stabilization of the pre-initiation complex by Mediator and suggest how Mediator might influence initiation by modulating polymerase conformation and interaction with promoter DNA.