RESUMO
The essential Mediator (MED) coactivator complex plays a well-understood role in regulation of basal transcription in all eukaryotes, but the mechanism underlying its role in activator-dependent transcription remains unknown. We investigated modulation of metazoan MED interaction with RNA polymerase II (RNA Pol II) by antagonistic effects of the MED26 subunit and the CDK8 kinase module (CKM). Biochemical analysis of CKM-MED showed that the CKM blocks binding of the RNA Pol II carboxy-terminal domain (CTD), preventing RNA Pol II interaction. This restriction is eliminated by nuclear receptor (NR) binding to CKM-MED, which enables CTD binding in a MED26-dependent manner. Cryoelectron microscopy (cryo-EM) and crosslinking-mass spectrometry (XL-MS) revealed that the structural basis for modulation of CTD interaction with MED relates to a large intrinsically disordered region (IDR) in CKM subunit MED13 that blocks MED26 and CTD interaction with MED but is repositioned upon NR binding. Hence, NRs can control transcription initiation by priming CKM-MED for MED26-dependent RNA Pol II interaction.
Assuntos
Microscopia Crioeletrônica , Quinase 8 Dependente de Ciclina , Complexo Mediador , Ligação Proteica , RNA Polimerase II , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Complexo Mediador/metabolismo , Complexo Mediador/genética , Complexo Mediador/química , Humanos , Quinase 8 Dependente de Ciclina/metabolismo , Quinase 8 Dependente de Ciclina/genética , Animais , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/química , Sítios de Ligação , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Células HEK293 , Domínios e Motivos de Interação entre ProteínasRESUMO
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
The potent HIV-1 capsid inhibitor GS-6207 is an investigational principal component of long-acting antiretroviral therapy. We found that GS-6207 inhibits HIV-1 by stabilizing and thereby preventing functional disassembly of the capsid shell in infected cells. X-ray crystallography, cryo-electron microscopy, and hydrogen-deuterium exchange experiments revealed that GS-6207 tightly binds two adjoining capsid subunits and promotes distal intra- and inter-hexamer interactions that stabilize the curved capsid lattice. In addition, GS-6207 interferes with capsid binding to the cellular HIV-1 cofactors Nup153 and CPSF6 that mediate viral nuclear import and direct integration into gene-rich regions of chromatin. These findings elucidate structural insights into the multimodal, potent antiviral activity of GS-6207 and provide a means for rationally developing second-generation therapies.
Assuntos
Fármacos Anti-HIV , Capsídeo , HIV-1 , Humanos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Capsídeo/química , Capsídeo/efeitos dos fármacos , Microscopia Crioeletrônica , Cristalografia por Raios X , Medição da Troca de Deutério , Células HEK293 , Células HeLa , HIV-1/química , HIV-1/efeitos dos fármacos , Fatores de Poliadenilação e Clivagem de mRNA/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Domínios Proteicos , Integração ViralRESUMO
While Mediator plays a key role in eukaryotic transcription, little is known about its mechanism of action. This study combines CRISPR-Cas9 genetic screens, degron assays, Hi-C, and cryoelectron microscopy (cryo-EM) to dissect the function and structure of mammalian Mediator (mMED). Deletion analyses in B, T, and embryonic stem cells (ESC) identified a core of essential subunits required for Pol II recruitment genome-wide. Conversely, loss of non-essential subunits mostly affects promoters linked to multiple enhancers. Contrary to current models, however, mMED and Pol II are dispensable to physically tether regulatory DNA, a topological activity requiring architectural proteins. Cryo-EM analysis revealed a conserved core, with non-essential subunits increasing structural complexity of the tail module, a primary transcription factor target. Changes in tail structure markedly increase Pol II and kinase module interactions. We propose that Mediator's structural pliability enables it to integrate and transmit regulatory signals and act as a functional, rather than an architectural bridge, between promoters and enhancers.
Assuntos
Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Proteínas Cromossômicas não Histona/metabolismo , Microscopia Crioeletrônica , Elementos Facilitadores Genéticos , Edição de Genes , Humanos , Masculino , Complexo Mediador/química , Complexo Mediador/genética , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Regiões Promotoras Genéticas , Estrutura Quaternária de Proteína , RNA Polimerase II/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , CoesinasRESUMO
Tight binding of Gdown1 represses RNA polymerase II (Pol II) function in a manner that is reversed by Mediator, but the structural basis of these processes is unclear. Although Gdown1 is intrinsically disordered, its Pol II interacting domains were localized and shown to occlude transcription factor IIF (TFIIF) and transcription factor IIB (TFIIB) binding by perfect positioning on their Pol II interaction sites. Robust binding of Gdown1 to Pol II is established by cooperative interactions of a strong Pol II binding region and two weaker binding modulatory regions, thus providing a mechanism both for tight Pol II binding and transcription inhibition and for its reversal. In support of a physiological function for Gdown1 in transcription repression, Gdown1 co-localizes with Pol II in transcriptionally silent nuclei of early Drosophila embryos but re-localizes to the cytoplasm during zygotic genome activation. Our study reveals a self-inactivation through Gdown1 binding as a unique mode of repression in Pol II function.
Assuntos
Regulação da Expressão Gênica , RNA Polimerase II/metabolismo , Transcrição Gênica , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Drosophila melanogaster/embriologia , Células HeLa , Humanos , Fatores de Transcrição TFII/genéticaRESUMO
Human adenoviruses (HAdVs) cause acute respiratory, ocular, and gastroenteric diseases and are also frequently used as gene and vaccine delivery vectors. Unlike the archetype human adenovirus C5 (HAdV-C5), human adenovirus D26 (HAdV-D26) belongs to species-D HAdVs, which target different cellular receptors, and is differentially recognized by immune surveillance mechanisms. HAdV-D26 is being championed as a lower seroprevalent vaccine and oncolytic vector in preclinical and human clinical studies. To understand the molecular basis for their distinct biological properties and independently validate the structures of minor proteins, we determined the first structure of species-D HAdV at 3.7 Å resolution by cryo-electron microscopy. All the hexon hypervariable regions (HVRs), including HVR1, have been identified and exhibit a distinct organization compared to those of HAdV-C5. Despite the differences in the arrangement of helices in the coiled-coil structures, protein IX molecules form a continuous hexagonal network on the capsid exterior. In addition to the structurally conserved region (3 to 300) of IIIa, we identified an extra helical domain comprising residues 314 to 390 that further stabilizes the vertex region. Multiple (two to three) copies of the cleaved amino-terminal fragment of protein VI (pVIn) are observed in each hexon cavity, suggesting that there could be ≥480 copies of VI present in HAdV-D26. In addition, a localized asymmetric reconstruction of the vertex region provides new details of the three-pronged "claw hold" of the trimeric fiber and its interactions with the penton base. These observations resolve the previous conflicting assignments of the minor proteins and suggest the likely conservation of their organization across different HAdVs.
Assuntos
Adenovírus Humanos/química , Proteínas do Capsídeo/química , Capsídeo/química , Microscopia Crioeletrônica/métodos , Humanos , Domínios ProteicosRESUMO
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
Mediator plays an integral role in activation of RNA polymerase II (Pol II) transcription. A key step in activation is binding of Mediator to Pol II to form the Mediator-Pol II holoenzyme. Here, we exploit a combination of biochemistry and macromolecular EM to investigate holoenzyme assembly. We identify a subset of human Mediator head module subunits that bind Pol II independent of other subunits and thus probably contribute to a major Pol II binding site. In addition, we show that binding of human Mediator to Pol II depends on the integrity of a conserved "hinge" in the middle module MED21-MED7 heterodimer. Point mutations in the hinge region leave core Mediator intact but lead to increased disorder of the middle module and markedly reduced affinity for Pol II. These findings highlight the importance of Mediator conformation for holoenzyme assembly.
Assuntos
Holoenzimas/metabolismo , Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Holoenzimas/química , Holoenzimas/genética , Humanos , Complexo Mediador/química , Complexo Mediador/genética , Ligação Proteica , Conformação Proteica , RNA Polimerase II/química , RNA Polimerase II/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
Ribonucleotide reductases (RNRs) are responsible for all de novo biosynthesis of DNA precursors in nature by catalyzing the conversion of ribonucleotides to deoxyribonucleotides. Because of its essential role in cell division, human RNR is a target for a number of anticancer drugs in clinical use. Like other class Ia RNRs, human RNR requires both a radical-generation subunit (ß) and nucleotide-binding subunit (α) for activity. Because of their complex dependence on allosteric effectors, however, the active and inactive quaternary forms of many class Ia RNRs have remained in question. Here, we present an X-ray crystal structure of the human α subunit in the presence of inhibiting levels of dATP, depicting a ring-shaped hexamer (α6) where the active sites line the inner hole. Surprisingly, our small-angle X-ray scattering (SAXS) results indicate that human α forms a similar hexamer in the presence of ATP, an activating effector. In both cases, α6 is assembled from dimers (α2) without a previously proposed tetramer intermediate (α4). However, we show with SAXS and electron microscopy that at millimolar ATP, the ATP-induced α6 can further interconvert with higher-order filaments. Differences in the dATP- and ATP-induced α6 were further examined by SAXS in the presence of the ß subunit and by activity assays as a function of ATP or dATP. Together, these results suggest that dATP-induced α6 is more stable than the ATP-induced α6 and that stabilization of this ring-shaped configuration provides a mechanism to prevent access of the ß subunit to the active site of α.
Assuntos
Nucleotídeos de Desoxiadenina/química , Nucleotídeos de Desoxiadenina/metabolismo , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Regulação Alostérica , Cristalografia por Raios X , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
The structure of a 33-protein, 1.5-MDa RNA polymerase II preinitiation complex (PIC) was determined by cryo-EM and image processing at a resolution of 6-11 Å. Atomic structures of over 50% of the mass were fitted into the electron density map in a manner consistent with protein-protein cross-links previously identified by mass spectrometry. The resulting model of the PIC confirmed the main conclusions from previous cryo-EM at lower resolution, including the association of promoter DNA only with general transcription factors and not with the polymerase. Electron density due to DNA was identifiable by the grooves of the double helix and exhibited sharp bends at points downstream of the TATA box, with an important consequence: The DNA at the downstream end coincides with the DNA in a transcribing polymerase. The structure of the PIC is therefore conducive to promoter melting, start-site scanning, and the initiation of transcription.
Assuntos
DNA/química , Complexos Multiproteicos/química , RNA Polimerase II/química , Fatores de Transcrição/química , Transcrição Gênica , Microscopia Crioeletrônica , DNA/genética , DNA/metabolismo , DNA/ultraestrutura , Humanos , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestrutura , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA Polimerase II/metabolismo , RNA Polimerase II/ultraestrutura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , TATA Box/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/ultraestrutura , Fatores de Transcrição TFII/química , Fatores de Transcrição TFII/metabolismo , Fatores de Transcrição TFII/ultraestruturaRESUMO
Natural proteins can be versatile building blocks for multimeric, self-assembling structures. Yet, creating protein-based assemblies with specific geometries and chemical properties remains challenging. Highly porous materials represent particularly interesting targets for designed assembly. Here, we utilize a strategy of fusing two natural protein oligomers using a continuous alpha-helical linker to design a novel protein that self assembles into a 750â kDa, 225â Å diameter, cube-shaped cage with large openings into a 130â Å diameter inner cavity. A crystal structure of the cage showed atomic-level agreement with the designed model, while electron microscopy, native mass spectrometry and small angle X-ray scattering revealed alternative assembly forms in solution. These studies show that accurate design of large porous assemblies with specific shapes is feasible, while further specificity improvements will probably require limiting flexibility to select against alternative forms. These results provide a foundation for the design of advanced materials with applications in bionanotechnology, nanomedicine and material sciences.
Assuntos
Proteínas/química , Cristalografia por Raios X , Peso Molecular , Porosidade , Espalhamento de RadiaçãoRESUMO
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
Designing protein molecules that self-assemble into complex architectures is an outstanding goal in the area of nanobiotechnology. One design strategy for doing this involves genetically fusing together two natural proteins, each of which is known to form a simple oligomer on its own (e.g., a dimer or trimer). If two such components can be fused in a geometrically predefined configuration, that designed subunit can, in principle, assemble into highly symmetric architectures. Initial experiments showed that a 12-subunit tetrahedral cage, 16 nm in diameter, could be constructed following such a procedure [Padilla, J. E.; et al. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 2217; Lai, Y. T.; et al. Science 2012, 336, 1129]. Here we characterize multiple crystal structures of protein cages constructed in this way, including cages assembled from two mutant forms of the same basic protein subunit. The flexibilities of the designed assemblies and their deviations from the target model are described, along with implications for further design developments.
Assuntos
Nanoestruturas/química , Proteínas/química , Cristalografia por Raios X , Modelos Moleculares , Mutação , Tamanho da Partícula , Conformação Proteica , Proteínas/genética , Propriedades de SuperfícieRESUMO
The CDK8 kinase module (CKM) is a conserved, dissociable Mediator subcomplex whose component subunits were genetically linked to the RNA polymerase II (RNAPII) C-terminal domain (CTD) and individually recognized as transcriptional repressors before Mediator was identified as a pre-eminent complex in eukaryotic transcription regulation. We used macromolecular EM and biochemistry to investigate the subunit organization, structure and Mediator interaction of the Saccharomyces cerevisiae CKM. We found that interaction of the CKM with Mediator's middle module interferes with CTD-dependent RNAPII binding to a previously unknown middle-module CTD-binding site and with the holoenzyme formation process. Taken together, our results reveal the basis for CKM repression, clarify the origin of the connection between CKM subunits and the CTD and suggest that a combination of competitive interactions and conformational changes that facilitate holoenzyme formation underlie the mechanism of transcription regulation by Mediator.
Assuntos
Quinase 8 Dependente de Ciclina/metabolismo , Regulação Fúngica da Expressão Gênica , Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Quinase 8 Dependente de Ciclina/química , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Complexo Mediador/química , Microscopia Eletrônica , Ligação Proteica , RNA Polimerase II/química , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates (dNDPs). The Escherichia coli class Ia RNR uses a mechanism of radical propagation by which a cysteine in the active site of the RNR large (α2) subunit is transiently oxidized by a stable tyrosyl radical (Yâ¢) in the RNR small (ß2) subunit over a 35-Å pathway of redox-active amino acids: Y122⢠â [W48?] â Y356 in ß2 to Y731 â Y730 â C439 in α2. When 3-aminotyrosine (NH2Y) is incorporated in place of Y730, a long-lived NH2Y730⢠is generated in α2 in the presence of wild-type (wt)-ß2, substrate, and effector. This radical intermediate is chemically and kinetically competent to generate dNDPs. Herein, evidence is presented that NH2Y730⢠induces formation of a kinetically stable α2ß2 complex. Under conditions that generate NH2Y730â¢, binding between Y730NH2Y-α2 and wt-ß2 is 25-fold tighter (Kd = 7 nM) than for wt-α2
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Domínio Catalítico , Transporte de Elétrons , Estabilidade Enzimática , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Cinética , Microscopia Eletrônica , Modelos Moleculares , Mutagênese Sítio-Dirigida , Estrutura Quaternária de Proteína , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleotídeo Redutases/classificação , Ribonucleotídeo Redutases/genética , Espalhamento a Baixo Ângulo , Espectrometria de Fluorescência , Difração de Raios XRESUMO
Clofarabine (ClF) is a drug used in the treatment of leukemia. One of its primary targets is human ribonucleotide reductase (hRNR), a dual-subunit, (α(2))(m)(ß(2))(n), regulatory enzyme indispensable in de novo dNTP synthesis. We report that, in live mammalian cells, ClF targets hRNR by converting its α-subunit into kinetically stable hexamers. We established mammalian expression platforms that enabled isolation of functional α and characterization of its altered oligomeric associations in response to ClF treatment. Size exclusion chromatography and electron microscopy documented persistence of in-cell-assembled-α(6). Our data validate hRNR as an important target of ClF, provide evidence that in vivo α's quaternary structure can be perturbed by a nonnatural ligand, and suggest small-molecule-promoted, persistent hexamerization as a strategy to modulate hRNR activity. These studies lay foundations for documentation of RNR oligomeric state within a cell.
Assuntos
Nucleotídeos de Adenina/farmacologia , Arabinonucleosídeos/farmacologia , Fígado/citologia , Fígado/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Subunidades Proteicas/antagonistas & inibidores , Ribonucleotídeo Redutases/antagonistas & inibidores , Ribonucleotídeo Redutases/química , Sobrevivência Celular , Clofarabina , Humanos , Cinética , Fígado/enzimologia , Estrutura Molecular , Conformação Proteica/efeitos dos fármacos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ribonucleotídeo Redutases/metabolismoRESUMO
Ribonucleotide reductases (RNRs) provide the precursors for DNA biosynthesis and repair and are successful targets for anticancer drugs such as clofarabine and gemcitabine. Recently, we reported that dATP inhibits E. coli class Ia RNR by driving formation of RNR subunits into α4ß4 rings. Here, we present the first X-ray structure of a gemcitabine-inhibited E. coli RNR and show that the previously described α4ß4 rings can interlock to form an unprecedented (α4ß4)2 megacomplex. This complex is also seen in a higher-resolution dATP-inhibited RNR structure presented here, which employs a distinct crystal lattice from that observed in the gemcitabine-inhibited case. With few reported examples of protein catenanes, we use data from small-angle X-ray scattering and electron microscopy to both understand the solution conditions that contribute to concatenation in RNRs as well as present a mechanism for the formation of these unusual structures.
Assuntos
Proteínas de Escherichia coli/química , Exorribonucleases/química , Cristalografia por Raios X , Cistina Difosfato/análogos & derivados , Cistina Difosfato/química , Nucleotídeos de Desoxiadenina/química , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/ultraestrutura , Exorribonucleases/antagonistas & inibidores , Exorribonucleases/ultraestrutura , Microscopia Eletrônica , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/química , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
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
Identification of homogeneous subsets of images in a macromolecular electron microscopy (EM) image data set is a critical step in single-particle analysis. The task is handled by iterative algorithms, whose performance is compromised by the compounded limitations of image alignment and K-means clustering. Here we describe an approach, iterative stable alignment and clustering (ISAC) that, relying on a new clustering method and on the concepts of stability and reproducibility, can extract validated, homogeneous subsets of images. ISAC requires only a small number of simple parameters and, with minimal human intervention, can eliminate bias from two-dimensional image clustering and maximize the quality of group averages that can be used for ab initio three-dimensional structural determination and analysis of macromolecular conformational variability. Repeated testing of the stability and reproducibility of a solution within ISAC eliminates heterogeneous or incorrect classes and introduces critical validation to the process of EM image clustering.