RESUMEN
Transcription initiation requires assembly of the RNA polymerase II (Pol II) pre-initiation complex (PIC) and opening of promoter DNA. Here, we present the long-sought high-resolution structure of the yeast PIC and define the mechanism of initial DNA opening. We trap the PIC in an intermediate state that contains half a turn of open DNA located 30-35 base pairs downstream of the TATA box. The initially opened DNA region is flanked and stabilized by the polymerase "clamp head loop" and the TFIIF "charged region" that both contribute to promoter-initiated transcription. TFIIE facilitates initiation by buttressing the clamp head loop and by regulating the TFIIH translocase. The initial DNA bubble is then extended in the upstream direction, leading to the open promoter complex and enabling start-site scanning and RNA synthesis. This unique mechanism of DNA opening may permit more intricate regulation than in the Pol I and Pol III systems.
Asunto(s)
ADN/química , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/metabolismo , Iniciación de la Transcripción Genética , Secuencia de Aminoácidos , Microscopía por Crioelectrón , ADN/ultraestructura , Modelos Biológicos , Modelos Moleculares , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , ARN Polimerasa II/ultraestructura , Eliminación de Secuencia , Factor de Transcripción TFIIH , Factores de Transcripción TFII/metabolismoRESUMEN
The initiation of transcription is a focal point for the regulation of gene activity during mammalian cell differentiation and development. To initiate transcription, RNA polymerase II (Pol II) assembles with general transcription factors into a pre-initiation complex (PIC) that opens promoter DNA. Previous work provided the molecular architecture of the yeast1-9 and human10,11 PIC and a topological model for DNA opening by the general transcription factor TFIIH12-14. Here we report the high-resolution cryo-electron microscopy structure of PIC comprising human general factors and Sus scrofa domesticus Pol II, which is 99.9% identical to human Pol II. We determine the structures of PIC with closed and opened promoter DNA at 2.5-2.8 Å resolution, and resolve the structure of TFIIH at 2.9-4.0 Å resolution. We capture the TFIIH translocase XPB in the pre- and post-translocation states, and show that XPB induces and propagates a DNA twist to initiate the opening of DNA approximately 30 base pairs downstream of the TATA box. We also provide evidence that DNA opening occurs in two steps and leads to the detachment of TFIIH from the core PIC, which may stop DNA twisting and enable RNA chain initiation.
Asunto(s)
ADN/química , ADN/metabolismo , Regiones Promotoras Genéticas/genética , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Animales , Emparejamiento Base , ADN/genética , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Mamíferos/genética , Modelos Moleculares , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , TATA Box/genética , Factor de Transcripción TFIIH/química , Factor de Transcripción TFIIH/metabolismo , Sitio de Iniciación de la Transcripción , Iniciación de la Transcripción GenéticaRESUMEN
Mediator is a conserved coactivator complex that enables the regulated initiation of transcription at eukaryotic genes1-3. Mediator is recruited by transcriptional activators and binds the pre-initiation complex (PIC) to stimulate the phosphorylation of RNA polymerase II (Pol II) and promoter escape1-6. Here we prepare a recombinant version of human Mediator, reconstitute a 50-subunit Mediator-PIC complex and determine the structure of the complex by cryo-electron microscopy. The head module of Mediator contacts the stalk of Pol II and the general transcription factors TFIIB and TFIIE, resembling the Mediator-PIC interactions observed in the corresponding complex in yeast7-9. The metazoan subunits MED27-MED30 associate with exposed regions in MED14 and MED17 to form the proximal part of the Mediator tail module that binds activators. Mediator positions the flexibly linked cyclin-dependent kinase (CDK)-activating kinase of the general transcription factor TFIIH near the linker to the C-terminal repeat domain of Pol II. The Mediator shoulder domain holds the CDK-activating kinase subunit CDK7, whereas the hook domain contacts a CDK7 element that flanks the kinase active site. The shoulder and hook domains reside in the Mediator head and middle modules, respectively, which can move relative to each other and may induce an active conformation of the CDK7 kinase to allosterically stimulate phosphorylation of the C-terminal domain.
Asunto(s)
Microscopía por Crioelectrón , Complejo Mediador/química , Complejo Mediador/ultraestructura , ARN Polimerasa II/química , ARN Polimerasa II/ultraestructura , Regulación Alostérica , Sitios de Unión , Dominio Catalítico , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/metabolismo , ADN Complementario/genética , Humanos , Complejo Mediador/metabolismo , Modelos Moleculares , Fosforilación , Unión Proteica , ARN Polimerasa II/metabolismo , Factor de Transcripción TFIIB/química , Factor de Transcripción TFIIB/metabolismo , Factores de Transcripción TFII/química , Factores de Transcripción TFII/metabolismo , Iniciación de la Transcripción Genética , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
Mitoribosomes consist of ribosomal RNA and protein components, coordinated assembly of which is critical for function. We used mitoribosomes from Trypanosoma brucei with reduced RNA and increased protein mass to provide insights into the biogenesis of the mitoribosomal large subunit. Structural characterization of a stable assembly intermediate revealed 22 assembly factors, some of which have orthologues/counterparts/homologues in mammalian genomes. These assembly factors form a protein network that spans a distance of 180 Å, shielding the ribosomal RNA surface. The central protuberance and L7/L12 stalk are not assembled entirely and require removal of assembly factors and remodeling of the mitoribosomal proteins to become functional. The conserved proteins GTPBP7 and mt-EngA are bound together at the subunit interface in proximity to the peptidyl transferase center. A mitochondrial acyl-carrier protein plays a role in docking the L1 stalk, which needs to be repositioned during maturation. Additional enzymatically deactivated factors scaffold the assembly while the exit tunnel is blocked. Together, this extensive network of accessory factors stabilizes the immature sites and connects the functionally important regions of the mitoribosomal large subunit.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Ribosomas Mitocondriales/metabolismo , Biosíntesis de Proteínas/fisiología , Subunidades Ribosómicas Grandes/metabolismo , Trypanosoma brucei brucei/metabolismo , Microscopía por Crioelectrón , Unión Proteica/fisiología , Conformación Proteica , ARN Ribosómico/genéticaRESUMEN
Human ALC1 is an oncogene-encoded chromatin-remodeling enzyme required for DNA repair that possesses a poly(ADP-ribose) (PAR)-binding macro domain. Its engagement with PARylated PARP1 activates ALC1 at sites of DNA damage, but the underlying mechanism remains unclear. Here, we establish a dual role for the macro domain in autoinhibition of ALC1 ATPase activity and coupling to nucleosome mobilization. In the absence of DNA damage, an inactive conformation of the ATPase is maintained by juxtaposition of the macro domain against predominantly the C-terminal ATPase lobe through conserved electrostatic interactions. Mutations within this interface displace the macro domain, constitutively activate the ALC1 ATPase independent of PARylated PARP1, and alter the dynamics of ALC1 recruitment at DNA damage sites. Upon DNA damage, binding of PARylated PARP1 by the macro domain induces a conformational change that relieves autoinhibitory interactions with the ATPase motor, which selectively activates ALC1 remodeling upon recruitment to sites of DNA damage.
Asunto(s)
Ensamble y Desensamble de Cromatina , Daño del ADN , ADN Helicasas/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Nucleosomas/enzimología , Dominio Catalítico , Línea Celular Tumoral , ADN Helicasas/química , ADN Helicasas/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Activación Enzimática , Humanos , Microscopía Electrónica , Simulación de Dinámica Molecular , Mutación , Nucleosomas/química , Poli(ADP-Ribosa) Polimerasa-1/química , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli ADP Ribosilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Dispersión del Ángulo Pequeño , Electricidad Estática , Relación Estructura-Actividad , Factores de Tiempo , Difracción de Rayos XRESUMEN
Eukaryotic gene transcription is carried out by three RNA polymerases: Pol I, Pol II and Pol III. Although it has long been known that Pol I can form homodimers, it is unclear whether and how the two other RNA polymerases dimerize. Here we present the cryo-electron microscopy (cryo-EM) structure of a mammalian Pol II dimer at 3.5 Å resolution. The structure differs from the Pol I dimer and reveals that one Pol II copy uses its RPB4-RPB7 stalk to penetrate the active centre cleft of the other copy, and vice versa, giving rise to a molecular handshake. The polymerase clamp domain is displaced and mobile, and the RPB7 oligonucleotide-binding fold mimics the DNA-RNA hybrid that occupies the cleft during active transcription. The Pol II dimer is incompatible with nucleic acid binding as required for transcription and may represent an inactive storage form of the polymerase.
Asunto(s)
ARN Polimerasa II/química , Animales , Microscopía por Crioelectrón , Dimerización , Modelos Moleculares , Multimerización de Proteína , Saccharomyces cerevisiae/enzimología , Sus scrofaRESUMEN
Transcription-export complex 2 (TREX-2, or THSC) facilitates localization of actively transcribing genes such as GAL1 to the nuclear periphery, contributes to the generation of export-competent mRNPs and influences gene expression through interactions with Mediator. TREX-2 is based on a Sac3 scaffold to which Thp1, Sem1, Cdc31 and Sus1 bind and consists of three modules: the N-region (Sac3â¼1-100), which binds mRNA export factor Mex67:Mtr2; the M-region, in which Thp1 and Sem1 bind to Sac3â¼100-550; and the CID region in which Cdc31 and two Sus1 chains bind to Sac3â¼720-805. Although the M-region of Sac3 was originally thought to encompass residues â¼250-550, we report here the 2.3Å resolution crystal structure of a complex containing Sac3 residues 60-550 that indicates that the TPR-like repeats of the M-region extend to residue 137 and that residues 90-125 form a novel loop that links Sac3 to Thp1. These new structural elements are important for growth and mRNA export in vivo. Although deleting Sac3 residues 1-90 produced a wild-type phenotype, deletion of the loop as well generated growth defects at 37°C, whereas the deletion of residues 1-250 impaired mRNA export and also generated longer lag times when glucose or raffinose was replaced by galactose as the carbon source.
Asunto(s)
Complejos Multiproteicos/metabolismo , ARN de Hongos/química , ARN de Hongos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Dominios Proteicos , Estructura Secundaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
In Saccharomyces cerevisiae generation of export-competent mRNPs terminates the nuclear phase of the gene expression pathway and facilitates transport to the cytoplasm for translation. Nab2 functions in this process to control both mRNP compaction that facilitates movement through nuclear pore complexes and the length of transcript poly(A) tails. Nab2 has a modular structure that includes seven CCCH Zn fingers that bind to A-rich RNAs and fingers 57 are critical for these functions. Here, we demonstrate, using both biophysical and structural methods, that binding A11G RNA induces dimerization of Zn fingers 57 mediated by the novel spatial arrangement of the fingers promoting each RNA chain binding two protein chains. The dimerization of Nab2 induced by RNA binding provides a basis for understanding its function in both poly(A) tail length regulation and in the compaction of mature transcripts to facilitate nuclear export.
Asunto(s)
Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transporte Activo de Núcleo Celular , Sustitución de Aminoácidos , Secuencia de Bases , Fenómenos Biofísicos , Cristalografía por Rayos X , Galactoquinasa/genética , Genes Fúngicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas de Transporte Nucleocitoplasmático/genética , Poliadenilación , Dominios Proteicos , Multimerización de Proteína , ARN de Hongos/química , ARN de Hongos/genética , ARN Mensajero/química , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Dedos de ZincRESUMEN
The anaphase-promoting complex/cyclosome (APC/C) is a large multimeric cullin-RING E3 ubiquitin ligase that orchestrates cell-cycle progression by targeting cell-cycle regulatory proteins for destruction via the ubiquitin proteasome system. The APC/C assembly comprises two scaffolding subcomplexes: the platform and the TPR lobe that together coordinate the juxtaposition of the catalytic and substrate-recognition modules. The platform comprises APC/C subunits Apc1, Apc4, Apc5, and Apc15. Although the role of Apc1 as an APC/C scaffolding subunit has been characterized, its specific functions in contributing toward APC/C catalytic activity are not fully understood. Here, we report the crystal structure of the N-terminal domain of human Apc1 (Apc1N) determined at 2.2-Å resolution and provide an atomic-resolution description of the architecture of its WD40 (WD40 repeat) domain (Apc1(WD40)). To understand how Apc1(WD40) contributes to APC/C activity, a mutant form of the APC/C with Apc1(WD40) deleted was generated and evaluated biochemically and structurally. We found that the deletion of Apc1(WD40) abolished the UbcH10-dependent ubiquitination of APC/C substrates without impairing the Ube2S-dependent ubiquitin chain elongation activity. A cryo-EM structure of an APC/C-Cdh1 complex with Apc1(WD40) deleted showed that the mutant APC/C is locked into an inactive conformation in which the UbcH10-binding site of the catalytic module is inaccessible. Additionally, an EM density for Apc15 is not visible. Our data show that Apc1(WD40) is required to mediate the coactivator-induced conformational change of the APC/C that is responsible for stimulating APC/C catalytic activity by promoting UbcH10 binding. In contrast, Ube2S activity toward APC/C substrates is not dependent on the initiation-competent conformation of the APC/C.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/química , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/química , Cadherinas/química , Proteínas de Ciclo Celular/química , Proteínas Mutantes/química , Regulación Alostérica/genética , Ciclosoma-Complejo Promotor de la Anafase/genética , Antígenos CD , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/genética , Sitios de Unión , Cadherinas/genética , Proteínas de Ciclo Celular/genética , Cristalografía por Rayos X , Humanos , Proteínas Mutantes/genética , Unión Proteica , Conformación Proteica , Dominios Proteicos , Ubiquitina/química , Ubiquitina/genética , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/genética , Repeticiones WD40/genéticaRESUMEN
The NXF1:NXT1 complex (also known as TAP:p15) is a general mRNA nuclear export factor that is conserved from yeast to humans. NXF1 is a modular protein constructed from four domains (RRM, LRR, NTF2-like and UBA domains). It is currently unclear how NXF1:NXT1 binds transcripts and whether there is higher organization of the NXF1 domains. We report here the 3.4 Å resolution crystal structure of the first three domains of human NXF1 together with NXT1 that has two copies of the complex in the asymmetric unit arranged to form an intimate domain-swapped dimer. In this dimer, the linkers between the NXF1 LRR and NTF2-like domains interact with NXT1, generating a 2-fold symmetric platform in which the RNA-binding RRM, LRR and NTF2-like domains are arranged on one face. In addition to bulk transcripts, NXF1:NXT1 also facilitates the export of unspliced retroviral genomic RNA from simple type-D retroviruses such as SRV-1 that contain a constitutive transport element (CTE), a cis-acting 2-fold symmetric RNA stem-loop motif. Complementary structural, biochemical and cellular techniques indicated that the formation of a symmetric RNA binding platform generated by dimerization of NXF1:NXT1 facilitates the recognition of CTE-RNA and promotes its nuclear export.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Retroviridae/genética , Transporte Biológico , Cristalografía por Rayos X , Dimerización , Células HEK293 , Humanos , Modelos Moleculares , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Unión al ARN/químicaRESUMEN
The Mex67:Mtr2 complex is the principal yeast nuclear export factor for bulk mRNA and also contributes to ribosomal subunit export. Mex67 is a modular protein constructed from four domains (RRM, LRR, NTF2-like and UBA) that have been thought to be joined by flexible linkers like beads on a string, with the RRM and LRR domains binding RNAs and the NTF2-like and UBA domains binding FG-nucleoporins to facilitate movement through nuclear pores. Here, we show that the NTF2-like domain from Saccharomyces cerevisiae Mex67:Mtr2 also contributes to RNA binding. Moreover, the 3.3 Å resolution crystal structure of the Mex67(ΔUBA):Mtr2 complex, supplemented with small angle X-ray scattering data, indicated that the LRR domain has a defined spatial relationship to the Mex67(NTF2L):Mtr2 region. Conversely, the RRM domain and especially the UBA domain are more mobile. The conformation assumed by the LRR and NTF2-like domains results in clusters of positively-charged residues on each becoming arranged to form a continuous interface for binding RNA on the opposite side of the complex to the region that interacts with FG-nucleoporins to facilitate passage through nuclear pores.
Asunto(s)
Proteínas de Transporte de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Proteínas de Transporte de Membrana/química , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas de Transporte Nucleocitoplasmático/química , Unión Proteica , Proteínas de Unión al ARN/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Dispersión de Radiación , Homología de Secuencia de AminoácidoRESUMEN
There is a lack of current treatment options for ovarian clear cell carcinoma (CCC) and the cancer is often resistant to platinum-based chemotherapy. Hence there is an urgent need for novel therapeutics. The transcription factor hepatocyte nuclear factor 1ß (HNF1ß) is ubiquitously overexpressed in CCC and is seen as an attractive therapeutic target. This was validated through shRNA-mediated knockdown of the target protein, HNF1ß, in five high- and low-HNF1ß-expressing CCC lines. To inhibit the protein function, cell-permeable, non-helical constrained proteomimetics to target the HNF1ß-importinâ α protein-protein interaction were designed, guided by X-ray crystallographic data and molecular dynamics simulations. In this way, we developed the first reported series of constrained peptide nuclear import inhibitors. Importantly, this general approach may be extended to other transcription factors.
Asunto(s)
Péptidos de Penetración Celular/metabolismo , Péptidos de Penetración Celular/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Péptidos de Penetración Celular/química , Cristalografía por Rayos X , Femenino , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Estructura Molecular , Neoplasias Ováricas/química , Unión Proteica/efectos de los fármacosRESUMEN
UNLABELLED: Transcription-export complex 2 (TREX-2 complex) facilitates the localization of actively transcribing genes to the nuclear periphery and also functions to contribute to the generation of export-competent mRNPs through interactions with the general mRNA nuclear export factor Mex67:Mtr2. The TREX-2 complex is based on a Sac3 scaffold to which Thp1, Sem1, Cdc31, and Sus1 bind. TREX-2 can be subdivided into two modules: one, in which Thp1 and Sem1 bind to the Sac3(M) region (residues â¼100-551), and the other in which Cdc31 and two Sus1 chains bind to the Sac3(CID) region (residues â¼710-805). Complementary structural analyses using X-ray crystallography, electron microscopy, and small-angle X-ray scattering of the Saccharomyces cerevisiae TREX-2 complex, expressed using Baculovirus-infected Sf9 cells, have indicated that the TPR-like repeats of the Sac3(M) region extend considerably further towards the N-terminus than previously thought, and also indicate that this region and Sac3(CID):Sus1:Cdc31 region of the S. cerevisiae complex are structurally independent. Although the density visible accounted for only â¼100kDa, a 5.3Å resolution cryo-EM reconstruction was obtained of the M-region of TREX-2 that showed an additional three putative α-helices extending towards the Sac3 N-terminus and these helices were also seen in a 4.9Å resolution structure obtained by X-ray crystallography. SUMMARY STATEMENT: We describe the expression, purification and structural characterization of the S. cerevisiae TREX-2 complex and demonstrate that the Sac3 TPR-like repeats are more extensive than previously thought and that the M- and CID-regions do not appear to have a defined spatial orientation.
Asunto(s)
Exodesoxirribonucleasas/química , Proteínas de Transporte Nucleocitoplasmático/química , Porinas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Animales , Microscopía por Crioelectrón , Cristalografía por Rayos X , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Dispersión del Ángulo Pequeño , Células Sf9 , SpodopteraRESUMEN
The transcription factor hepatocyte nuclear factor 1ß (HNF1ß) is ubiquitously overexpressed in ovarian clear cell carcinoma (CCC) and is a potential therapeutic target. To explore potential approaches that block HNF1ß transcription we have identified and characterised extensively the nuclear localisation signal (NLS) for HNF1ß and its interactions with the nuclear protein import receptor, Importin-α. Pull-down assays demonstrated that the DNA binding domain of HNF1ß interacted with a spectrum of Importin-α isoforms and deletion constructs tagged with eGFP confirmed that the HNF1ß (229)KKMRRNR(235) sequence was essential for nuclear localisation. We further characterised the interaction between the NLS and Importin-α using complementary biophysical techniques and have determined the 2.4Å resolution crystal structure of the HNF1ß NLS peptide bound to Importin-α. The functional, biochemical, and structural characterisation of the nuclear localisation signal present on HNF1ß and its interaction with the nuclear import protein Importin-α provide the basis for the development of compounds targeting transcription factor HNF1ß via its nuclear import pathway.
Asunto(s)
Núcleo Celular/metabolismo , Factor Nuclear 1-beta del Hepatocito/metabolismo , Transporte Activo de Núcleo Celular , Animales , Sitios de Unión , Línea Celular Tumoral , Cristalografía por Rayos X , Células HEK293 , Factor Nuclear 1-beta del Hepatocito/química , Humanos , Ratones , Modelos Moleculares , Señales de Localización Nuclear , Unión Proteica , Conformación Proteica en Hélice alfa , Análisis de Secuencia de Proteína , Proteínas de Xenopus/química , Xenopus laevis , alfa Carioferinas/químicaRESUMEN
The mitoribosome translates mitochondrial mRNAs and regulates energy conversion that is a signature of aerobic life forms. We present a 2.2 Å resolution structure of human mitoribosome together with validated mitoribosomal RNA (rRNA) modifications, including aminoacylated CP-tRNAVal. The structure shows how mitoribosomal proteins stabilise binding of mRNA and tRNA helping to align it in the decoding center, whereas the GDP-bound mS29 stabilizes intersubunit communication. Comparison between different states, with respect to tRNA position, allowed us to characterize a non-canonical L1 stalk, and molecular dynamics simulations revealed how it facilitates tRNA transitions in a way that does not require interactions with rRNA. We also report functionally important polyamines that are depleted when cells are subjected to an antibiotic treatment. The structural, biochemical, and computational data illuminate the principal functional components of the translation mechanism in mitochondria and provide a description of the structure and function of the human mitoribosome.
Asunto(s)
Ribosomas Mitocondriales , ARN de Transferencia , Humanos , ARN de Transferencia/metabolismo , ARN de Transferencia/química , ARN de Transferencia/genética , Ribosomas Mitocondriales/metabolismo , Ribosomas Mitocondriales/química , Ligandos , Simulación de Dinámica Molecular , ARN Mensajero/metabolismo , ARN Mensajero/genética , Mitocondrias/metabolismo , ARN Ribosómico/metabolismo , ARN Ribosómico/química , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/química , Guanosina Difosfato/metabolismo , Poliaminas/metabolismo , Poliaminas/química , Unión ProteicaRESUMEN
The mitoribosome translates mitochondrial mRNAs and regulates energy conversion that is a signature of aerobic life forms. We present a 2.2 Å resolution structure of human mitoribosome together with validated mitoribosomal RNA (rRNA) modifications, including aminoacylated CP-tRNA Val . The structure shows how mitoribosomal proteins stabilise binding of mRNA and tRNA helping to align it in the decoding center, whereas the GDP-bound mS29 stabilizes intersubunit communication. Comparison between different states, with respect to tRNA position, allowed to characterize a non-canonical L1 stalk, and molecular dynamics simulations revealed how it facilitates tRNA transition in a way that does not require interactions with rRNA. We also report functionally important polyamines that are depleted when cells are subjected to an antibiotic treatment. The structural, biochemical, and computational data illuminate the principal functional components of the translation mechanism in mitochondria and provide the most complete description so far of the structure and function of the human mitoribosome.
RESUMEN
Nuclear import of RNA polymerase II (Pol II) involves the conserved factor RPAP2. Here we report the cryo-electron microscopy (cryo-EM) structure of mammalian Pol II in complex with human RPAP2 at 2.8 Å resolution. The structure shows that RPAP2 binds between the jaw domains of the polymerase subunits RPB1 and RPB5. RPAP2 is incompatible with binding of downstream DNA during transcription and is displaced upon formation of a transcription pre-initiation complex.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Microscopía por Crioelectrón/métodos , ADN/metabolismo , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Transcripción Genética , Animales , Proteínas Portadoras/genética , Humanos , Mamíferos , Conformación Proteica , ARN Polimerasa II/genéticaRESUMEN
To initiate cotranscriptional splicing, RNA polymerase II (Pol II) recruits the U1 small nuclear ribonucleoprotein particle (U1 snRNP) to nascent precursor messenger RNA (pre-mRNA). Here, we report the cryo-electron microscopy structure of a mammalian transcribing Pol II-U1 snRNP complex. The structure reveals that Pol II and U1 snRNP interact directly. This interaction positions the pre-mRNA 5' splice site near the RNA exit site of Pol II. Extension of pre-mRNA retains the 5' splice site, leading to the formation of a "growing intron loop." Loop formation may facilitate scanning of nascent pre-mRNA for the 3' splice site, functional pairing of distant intron ends, and prespliceosome assembly. Our results provide a starting point for a mechanistic analysis of cotranscriptional spliceosome assembly and the biogenesis of mRNA isoforms by alternative splicing.
Asunto(s)
Empalme Alternativo , ARN Polimerasa II/química , ARN Mensajero/biosíntesis , Ribonucleoproteína Nuclear Pequeña U1/química , Empalmosomas/química , Animales , Microscopía por Crioelectrón , Humanos , Intrones , Conformación de Ácido Nucleico , Unión Proteica , Dominios Proteicos , Precursores del ARN/química , ARN Mensajero/química , Empalmosomas/metabolismo , Sus scrofa , Transcripción GenéticaRESUMEN
Glioblastoma stem cells (GSCs) resist current glioblastoma (GBM) therapies. GSCs rely highly on oxidative phosphorylation (OXPHOS), whose function requires mitochondrial translation. Here we explore the therapeutic potential of targeting mitochondrial translation and report the results of high-content screening with putative blockers of mitochondrial ribosomes. We identify the bacterial antibiotic quinupristin/dalfopristin (Q/D) as an effective suppressor of GSC growth. Q/D also decreases the clonogenicity of GSCs in vitro, consequently dysregulating the cell cycle and inducing apoptosis. Cryoelectron microscopy (cryo-EM) reveals that Q/D binds to the large mitoribosomal subunit, inhibiting mitochondrial protein synthesis and functionally dysregulating OXPHOS complexes. These data suggest that targeting mitochondrial translation could be explored to therapeutically suppress GSC growth in GBM and that Q/D could potentially be repurposed for cancer treatment.
Asunto(s)
Glioblastoma/genética , Mitocondrias/metabolismo , Células Madre Neoplásicas/metabolismo , Línea Celular Tumoral , Proliferación Celular , HumanosRESUMEN
Translation of mitochondrial messenger RNA (mt-mRNA) is performed by distinct mitoribosomes comprising at least 36 mitochondria-specific proteins. How these mitoribosomal proteins assist in the binding of mt-mRNA and to what extent they are involved in the translocation of transfer RNA (mt-tRNA) is unclear. To visualize the process of translation in human mitochondria, we report ~3.0 Å resolution structure of the human mitoribosome, including the L7/L12 stalk, and eight structures of its functional complexes with mt-mRNA, mt-tRNAs, recycling factor and additional trans factors. The study reveals a transacting protein module LRPPRC-SLIRP that delivers mt-mRNA to the mitoribosomal small subunit through a dedicated platform formed by the mitochondria-specific protein mS39. Mitoribosomal proteins of the large subunit mL40, mL48, and mL64 coordinate translocation of mt-tRNA. The comparison between those structures shows dynamic interactions between the mitoribosome and its ligands, suggesting a sequential mechanism of conformational changes.