RESUMEN
Eukaryotic ribosome biogenesis depends on several hundred assembly factors to produce functional 40S and 60S ribosomal subunits. The final phase of 60S subunit biogenesis is cytoplasmic maturation, which includes the proofreading of functional centers of the 60S subunit and the release of several ribosome biogenesis factors. We report the cryo-electron microscopy (cryo-EM) structure of the yeast 60S subunit in complex with the biogenesis factors Rei1, Arx1, and Alb1 at 3.4 Å resolution. In addition to the network of interactions formed by Alb1, the structure reveals a mechanism for ensuring the integrity of the ribosomal polypeptide exit tunnel. Arx1 probes the entire set of inner-ring proteins surrounding the tunnel exit, and the C terminus of Rei1 is deeply inserted into the ribosomal tunnel, where it forms specific contacts along almost its entire length. We provide genetic and biochemical evidence that failure to insert the C terminus of Rei1 precludes subsequent steps of 60S maturation.
Asunto(s)
Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Chaetomium/metabolismo , Microscopía por Crioelectrón , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Humanos , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/ultraestructura , Subunidades Ribosómicas Grandes de Eucariotas/ultraestructura , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestructura , Alineación de SecuenciaRESUMEN
Nonstructural protein 1 (Nsp1) produced by coronaviruses inhibits host protein synthesis. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Nsp1 C-terminal domain was shown to bind the ribosomal mRNA channel to inhibit translation, but it is unclear whether this mechanism is broadly used by coronaviruses, whether the Nsp1 N-terminal domain binds the ribosome, or how Nsp1 allows viral RNAs to be translated. Here, we investigated Nsp1 from SARS-CoV-2, Middle East respiratory syndrome coronavirus (MERS-CoV), and Bat-Hp-CoV coronaviruses using structural, biophysical, and biochemical experiments, revealing a conserved role for the C-terminal domain. Additionally, the N-terminal domain of Bat-Hp-CoV Nsp1 binds to the decoding center of the 40S subunit, where it would prevent mRNA and eIF1A accommodation. Structure-based experiments demonstrated the importance of decoding center interactions in all three coronaviruses and showed that the same regions of Nsp1 are necessary for the selective translation of viral RNAs. Our results provide a mechanistic framework to understand how Nsp1 controls preferential translation of viral RNAs.
Asunto(s)
COVID-19 , Quirópteros , Animales , Quirópteros/genética , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Dominios Proteicos , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
In contrast to the bacterial translation machinery, mitoribosomes and mitochondrial translation factors are highly divergent in terms of composition and architecture. There is increasing evidence that the biogenesis of mitoribosomes is an intricate pathway, involving many assembly factors. To better understand this process, we investigated native assembly intermediates of the mitoribosomal large subunit from the human parasite Trypanosoma brucei using cryo-electron microscopy. We identify 28 assembly factors, 6 of which are homologous to bacterial and eukaryotic ribosome assembly factors. They interact with the partially folded rRNA by specifically recognizing functionally important regions such as the peptidyltransferase center. The architectural and compositional comparison of the assembly intermediates indicates a stepwise modular assembly process, during which the rRNA folds toward its mature state. During the process, several conserved GTPases and a helicase form highly intertwined interaction networks that stabilize distinct assembly intermediates. The presented structures provide general insights into mitoribosomal maturation.
Asunto(s)
Ribosomas Mitocondriales/química , ARN Ribosómico/metabolismo , Subunidades Ribosómicas Grandes/química , Trypanosoma brucei brucei/metabolismo , Microscopía por Crioelectrón , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/metabolismo , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Ribosomas Mitocondriales/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , ARN Ribosómico/química , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Grandes/metabolismo , Trypanosoma brucei brucei/genéticaRESUMEN
After having translated short upstream open reading frames, ribosomes can re-initiate translation on the same mRNA. This process, referred to as re-initiation, controls the translation of a large fraction of mammalian cellular mRNAs, many of which are important in cancer. Key ribosomal binding proteins involved in re-initiation are the eukaryotic translation initiation factor 2D (eIF2D) or the homologous complex of MCT-1/DENR. We determined the structures of these factors bound to the human 40S ribosomal subunit in complex with initiator tRNA positioned on an mRNA start codon in the P-site using a combination of cryoelectron microscopy and X-ray crystallography. The structures, supported by biochemical experiments, reveal how eIF2D emulates the function of several canonical translation initiation factors by using three independent, flexibly connected RNA binding domains to simultaneously monitor codon-anticodon interactions in the ribosomal P-site and position the initiator tRNA.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Proteínas Oncogénicas/metabolismo , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Sitio de Iniciación de la Transcripción , Iniciación de la Transcripción Genética , Sitios de Unión , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Microscopía por Crioelectrón , Cristalografía por Rayos X , Factor 2 Eucariótico de Iniciación/química , Factor 2 Eucariótico de Iniciación/genética , Factores Eucarióticos de Iniciación/química , Factores Eucarióticos de Iniciación/genética , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Complejos Multiproteicos , Mutación , Conformación de Ácido Nucleico , Proteínas Oncogénicas/química , Proteínas Oncogénicas/genética , Unión Proteica , Conformación Proteica , ARN Mensajero/química , ARN Mensajero/genética , ARN de Transferencia/química , ARN de Transferencia/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/química , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Relación Estructura-Actividad , TransfecciónRESUMEN
Mitochondrial ribosomes (mitoribosomes) play a central role in synthesizing mitochondrial inner membrane proteins responsible for oxidative phosphorylation. Although mitoribosomes from different organisms exhibit considerable structural variations, recent insights into mitoribosome assembly suggest that mitoribosome maturation follows common principles and involves a number of conserved assembly factors. To investigate the steps involved in the assembly of the mitoribosomal small subunit (mt-SSU) we determined the cryoelectron microscopy structures of middle and late assembly intermediates of the Trypanosoma brucei mitochondrial small subunit (mt-SSU) at 3.6- and 3.7-Å resolution, respectively. We identified five additional assembly factors that together with the mitochondrial initiation factor 2 (mt-IF-2) specifically interact with functionally important regions of the rRNA, including the decoding center, thereby preventing premature mRNA or large subunit binding. Structural comparison of assembly intermediates with mature mt-SSU combined with RNAi experiments suggests a noncanonical role of mt-IF-2 and a stepwise assembly process, where modular exchange of ribosomal proteins and assembly factors together with mt-IF-2 ensure proper 9S rRNA folding and protein maturation during the final steps of assembly.
Asunto(s)
Proteínas Mitocondriales/química , Ribosomas Mitocondriales/química , Fosforilación Oxidativa , ARN Ribosómico/química , Proteínas Ribosómicas/química , Subunidades Ribosómicas/química , Línea Celular , Microscopía por Crioelectrón , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Ribosomas Mitocondriales/metabolismo , Modelos Moleculares , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas/genética , Subunidades Ribosómicas/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMEN
Mitochondria maintain their own specialized protein synthesis machinery, which in mammals is used exclusively for the synthesis of the membrane proteins responsible for oxidative phosphorylation1,2. The initiation of protein synthesis in mitochondria differs substantially from bacterial or cytosolic translation systems. Mitochondrial translation initiation lacks initiation factor 1, which is essential in all other translation systems from bacteria to mammals3,4. Furthermore, only one type of methionyl transfer RNA (tRNAMet) is used for both initiation and elongation4,5, necessitating that the initiation factor specifically recognizes the formylated version of tRNAMet (fMet-tRNAMet). Lastly, most mitochondrial mRNAs do not possess 5' leader sequences to promote mRNA binding to the ribosome2. There is currently little mechanistic insight into mammalian mitochondrial translation initiation, and it is not clear how mRNA engagement, initiator-tRNA recruitment and start-codon selection occur. Here we determine the cryo-EM structure of the complete translation initiation complex from mammalian mitochondria at 3.2 Å. We describe the function of an additional domain insertion that is present in the mammalian mitochondrial initiation factor 2 (mtIF2). By closing the decoding centre, this insertion stabilizes the binding of leaderless mRNAs and induces conformational changes in the rRNA nucleotides involved in decoding. We identify unique features of mtIF2 that are required for specific recognition of fMet-tRNAMet and regulation of its GTPase activity. Finally, we observe that the ribosomal tunnel in the initiating ribosome is blocked by insertion of the N-terminal portion of mitochondrial protein mL45, which becomes exposed as the ribosome switches to elongation mode and may have an additional role in targeting of mitochondrial ribosomes to the protein-conducting pore in the inner mitochondrial membrane.
Asunto(s)
Microscopía por Crioelectrón , Mamíferos , Mitocondrias/ultraestructura , Iniciación de la Cadena Peptídica Traduccional , Animales , Codón Iniciador/genética , Factores Eucarióticos de Iniciación/química , Factores Eucarióticos de Iniciación/genética , Factores Eucarióticos de Iniciación/metabolismo , Factores Eucarióticos de Iniciación/ultraestructura , Mitocondrias/química , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/ultraestructura , Modelos Moleculares , ARN Mitocondrial/química , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo , ARN Mitocondrial/ultraestructura , ARN de Transferencia de Metionina/genética , ARN de Transferencia de Metionina/metabolismo , ARN de Transferencia de Metionina/ultraestructuraRESUMEN
Final maturation of eukaryotic ribosomes occurs in the cytoplasm and requires the sequential removal of associated assembly factors and processing of the immature 20S pre-RNA Using cryo-electron microscopy (cryo-EM), we have determined the structure of a yeast cytoplasmic pre-40S particle in complex with Enp1, Ltv1, Rio2, Tsr1, and Pno1 assembly factors poised to initiate final maturation. The structure reveals that the pre-rRNA adopts a highly distorted conformation of its 3' major and 3' minor domains stabilized by the binding of the assembly factors. This observation is consistent with a mechanism that involves concerted release of the assembly factors orchestrated by the folding of the rRNA in the head of the pre-40S subunit during the final stages of maturation. Our results provide a structural framework for the coordination of the final maturation events that drive a pre-40S particle toward the mature form capable of engaging in translation.
Asunto(s)
Microscopía por Crioelectrón , Simulación del Acoplamiento Molecular , Proteínas Ribosómicas/ultraestructura , Subunidades Ribosómicas Pequeñas de Eucariotas/ultraestructura , Proteínas de Saccharomyces cerevisiae/ultraestructura , Saccharomyces cerevisiae/ultraestructura , Citoplasma , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/ultraestructura , Conformación Proteica , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/ultraestructura , Pliegue del ARN , ARN Ribosómico/química , ARN Ribosómico/ultraestructura , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/ultraestructura , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/aislamiento & purificación , Subunidades Ribosómicas Pequeñas de Eucariotas/química , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificaciónRESUMEN
Chloroplasts are cellular organelles of plants and algae that are responsible for energy conversion and carbon fixation by the photosynthetic reaction. As a consequence of their endosymbiotic origin, they still contain their own genome and the machinery for protein biosynthesis. Here, we present the atomic structure of the chloroplast 70S ribosome prepared from spinach leaves and resolved by cryo-EM at 3.4 Å resolution. The complete structure reveals the features of the 4.5S rRNA, which probably evolved by the fragmentation of the 23S rRNA, and all five plastid-specific ribosomal proteins. These proteins, required for proper assembly and function of the chloroplast translation machinery, bind and stabilize rRNA including regions that only exist in the chloroplast ribosome. Furthermore, the structure reveals plastid-specific extensions of ribosomal proteins that extensively remodel the mRNA entry and exit site on the small subunit as well as the polypeptide tunnel exit and the putative binding site of the signal recognition particle on the large subunit. The translation factor pY, involved in light- and temperature-dependent control of protein synthesis, is bound to the mRNA channel of the small subunit and interacts with 16S rRNA nucleotides at the A-site and P-site, where it protects the decoding centre and inhibits translation by preventing tRNA binding. The small subunit is locked by pY in a non-rotated state, in which the intersubunit bridges to the large subunit are stabilized.
Asunto(s)
Cloroplastos , Ribosomas/química , Ribosomas/ultraestructura , Spinacia oleracea , Microscopía por Crioelectrón , Modelos Moleculares , ARN Ribosómico/química , ARN Ribosómico/ultraestructura , Proteínas Ribosómicas/química , Proteínas Ribosómicas/ultraestructuraRESUMEN
Mitochondrial ribosomes (mitoribosomes) are extensively modified ribosomes of bacterial descent specialized for the synthesis and insertion of membrane proteins that are critical for energy conversion and ATP production inside mitochondria. Mammalian mitoribosomes, which comprise 39S and 28S subunits, have diverged markedly from the bacterial ribosomes from which they are derived, rendering them unique compared to bacterial, eukaryotic cytosolic and fungal mitochondrial ribosomes. We have previously determined at 4.9 Å resolution the architecture of the porcine (Sus scrofa) 39S subunit, which is highly homologous to the human mitoribosomal large subunit. Here we present the complete atomic structure of the porcine 39S large mitoribosomal subunit determined in the context of a stalled translating mitoribosome at 3.4 Å resolution by cryo-electron microscopy and chemical crosslinking/mass spectrometry. The structure reveals the locations and the detailed folds of 50 mitoribosomal proteins, shows the highly conserved mitoribosomal peptidyl transferase active site in complex with its substrate transfer RNAs, and defines the path of the nascent chain in mammalian mitoribosomes along their idiosyncratic exit tunnel. Furthermore, we present evidence that a mitochondrial tRNA has become an integral component of the central protuberance of the 39S subunit where it architecturally substitutes for the absence of the 5S ribosomal RNA, a ubiquitous component of all cytoplasmic ribosomes.
Asunto(s)
Mitocondrias/química , Proteínas Mitocondriales/química , Proteínas Mitocondriales/ultraestructura , Subunidades Ribosómicas Grandes/química , Subunidades Ribosómicas Grandes/ultraestructura , Animales , Reactivos de Enlaces Cruzados , Microscopía por Crioelectrón , Espectrometría de Masas , Mitocondrias/ultraestructura , Proteínas Mitocondriales/metabolismo , Modelos Moleculares , Conformación Molecular , Peptidil Transferasas/metabolismo , ARN Ribosómico/química , ARN Ribosómico/metabolismo , ARN Ribosómico/ultraestructura , Subunidades Ribosómicas Grandes/genética , Sus scrofa/genéticaRESUMEN
Mitochondrial ribosomes synthesize a number of highly hydrophobic proteins encoded on the genome of mitochondria, the organelles in eukaryotic cells that are responsible for energy conversion by oxidative phosphorylation. The ribosomes in mammalian mitochondria have undergone massive structural changes throughout their evolution, including ribosomal RNA shortening and acquisition of mitochondria-specific ribosomal proteins. Here we present the three-dimensional structure of the 39S large subunit of the porcine mitochondrial ribosome determined by cryo-electron microscopy at 4.9 Å resolution. The structure, combined with data from chemical crosslinking and mass spectrometry experiments, reveals the unique features of the 39S subunit at near-atomic resolution and provides detailed insight into the architecture of the polypeptide exit site. This region of the mitochondrial ribosome has been considerably remodelled compared to its bacterial counterpart, providing a specialized platform for the synthesis and membrane insertion of the highly hydrophobic protein components of the respiratory chain.
Asunto(s)
Mitocondrias/química , Subunidades Ribosómicas/química , Animales , Bovinos , Microscopía por Crioelectrón , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de Masas , Mitocondrias/ultraestructura , Proteínas Mitocondriales/química , Proteínas Mitocondriales/ultraestructura , Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica , ARN Ribosómico 16S/química , ARN Ribosómico 16S/ultraestructura , Proteínas Ribosómicas/química , Proteínas Ribosómicas/ultraestructura , Subunidades Ribosómicas/ultraestructura , PorcinosRESUMEN
Analogous to eukaryotic ubiquitination, proteins in actinobacteria can be post-translationally modified in a process referred to as pupylation, the covalent attachment of prokaryotic ubiquitin-like protein Pup to lysine side chains of the target protein via an isopeptide bond. As in eukaryotes, an opposing activity counteracts the modification by specific cleavage of the isopeptide bond formed with Pup. However, the enzymes involved in pupylation and depupylation have evolved independently of ubiquitination and are related to the family of ATP-binding and hydrolyzing carboxylate-amine ligases of the glutamine synthetase type. Furthermore, the Pup ligase PafA and the depupylase Dop share close structural and sequence homology and have a common evolutionary history despite catalyzing opposing reactions. Here, we investigate the role played by the nucleotide in the active site of the depupylase Dop using a combination of biochemical experiments and X-ray crystallographic studies. We show that, although Dop does not turn over ATP stoichiometrically with substrate, the active site nucleotide species in Dop is ADP and inorganic phosphate rather than ATP, and that non-hydrolyzable analogs of ATP cannot support the enzymatic reaction. This finding suggests that the catalytic mechanism is more similar to the mechanism of the ligase PafA than previously thought and likely involves the transient formation of a phosphorylated Pup-intermediate. Evidence is presented for a mechanism where the inorganic phosphate acts as the nucleophilic species in amide bond cleavage and implications for Dop function are discussed.
Asunto(s)
Actinobacteria/metabolismo , Amidohidrolasas/química , Amidohidrolasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Ligasas/metabolismo , Fosfatos/metabolismo , Actinobacteria/crecimiento & desarrollo , Adenosina Trifosfato/metabolismo , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Hidrólisis , Lisina/metabolismo , Procesamiento Proteico-Postraduccional , Ubiquitina/metabolismoRESUMEN
Eukaryotic ribosomes are significantly larger and more complex than their prokaryotic counterparts. This parallels the increased complexity of the associated cellular machinery responsible for translation initiation, ribosome assembly, and the regulation of protein synthesis in eukaryotic cells. The recently determined crystal structures of the small (40S) and large (60S) ribosomal subunits and the 80S ribosome now provide an atomic description of this essential molecular machine and reveal its eukaryote-specific features. In this review, we discuss the common structural principles underlying the evolution of both ribosomal subunits. The recently obtained structural information provides a framework for further genetic, biochemical and structural studies of eukaryotic ribosomes. At the same time, it facilitates a direct comparison between prokaryotic and eukaryotic ribosomal features.
Asunto(s)
Biosíntesis de Proteínas , ARN Ribosómico/química , Proteínas Ribosómicas/química , Ribosomas/química , Cristalografía por Rayos X , Células Eucariotas/metabolismo , Modelos Moleculares , ARN Ribosómico/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismoRESUMEN
Acyl carrier protein (ACP) domains are critical integral components of multifunctional type I fatty acid synthases (FAS I) and polyketide synthases (PKSs), where they shuttle the growing adducts of the synthesis between the catalytic domains. In contrast to ACP of mammalian FAS I, PKSs, and the dissociated fatty acid synthase type II systems (FAS II) of bacteria, fungal FAS I ACP consists of two subdomains, one comprising the canonical ACP fold observed in all FAS systems and the other representing an extra structural subdomain. While ACPs of dissociated FAS II are able to sequester the reaction intermediates during substrate shuttling, such a transport mechanism has not been observed in ACP domains of multifunctional FAS I and PKS systems. For a better understanding of the interaction between the canonical subdomain of fungal ACP with the growing acyl chain and the role of the structural subdomain, we determined the structure of the isolated Saccharomyces cerevisiae acyl carrier protein (ScACP) domain by NMR spectroscopy and investigated the interactions between ScACP and covalently attached substrate acyl chains of varying length by monitoring chemical shift perturbations. The interactions were mapped to the hydrophobic core of the canonical subdomain, while no perturbations were detected in the structural subdomain. A population analysis revealed that only approximately 15% of covalently attached decanoyl chains are sequestered by the ACP core, comparable to the mammalian FAS I and multifunctional PKS systems, which do not sequester their substrates. Finally, denaturation experiments show that both ScACP subdomains unfold cooperatively and that the weak interaction of the acyl chain with the hydrophobic core does not significantly affect the ACP stability.
Asunto(s)
Proteína Transportadora de Acilo/química , Proteína Transportadora de Acilo/metabolismo , Acido Graso Sintasa Tipo II/química , Acido Graso Sintasa Tipo I/química , Proteínas de Saccharomyces cerevisiae/química , Acido Graso Sintasa Tipo I/metabolismo , Acido Graso Sintasa Tipo II/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The translocon-associated protein (TRAP) complex resides in the endoplasmic reticulum (ER) membrane and interacts with the Sec translocon and the ribosome to facilitate biogenesis of secretory and membrane proteins. TRAP plays a key role in the secretion of many hormones, including insulin. Here we reveal the molecular architecture of the mammalian TRAP complex and how it engages the translating ribosome associated with Sec61 translocon on the ER membrane. The TRAP complex is anchored to the ribosome via a long tether and its position is further stabilized by a finger-like loop. This positions a cradle-like lumenal domain of TRAP below the translocon for interactions with translocated nascent chains. Our structure-guided TRAP mutations in Caenorhabditis elegans lead to growth deficits associated with increased ER stress and defects in protein hormone secretion. These findings elucidate the molecular basis of the TRAP complex in the biogenesis and translocation of proteins at the ER.
Asunto(s)
Retículo Endoplásmico , Glicoproteínas de Membrana , Animales , Glicoproteínas de Membrana/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Canales de Translocación SEC/metabolismo , Transporte de Proteínas , Mamíferos/metabolismoRESUMEN
Nonstructural protein 1 (Nsp1) produced by coronaviruses shuts down host protein synthesis in infected cells. The C-terminal domain of SARS-CoV-2 Nsp1 was shown to bind to the small ribosomal subunit to inhibit translation, but it is not clear whether this mechanism is broadly used by coronaviruses, whether the N-terminal domain of Nsp1 binds the ribosome, or how Nsp1 specifically permits translation of viral mRNAs. Here, we investigated Nsp1 from three representative Betacoronaviruses - SARS-CoV-2, MERS-CoV, and Bat-Hp-CoV - using structural, biophysical, and biochemical assays. We revealed a conserved mechanism of host translational shutdown across the three coronaviruses. We further demonstrated that the N-terminal domain of Bat-Hp-CoV Nsp1 binds to the decoding center of the 40S subunit, where it would prevent mRNA and eIF1A binding. Structure-based biochemical experiments identified a conserved role of these inhibitory interactions in all three coronaviruses and showed that the same regions of Nsp1 are responsible for the preferential translation of viral mRNAs. Our results provide a mechanistic framework to understand how Betacoronaviruses overcome translational inhibition to produce viral proteins.
RESUMEN
The genetic code that specifies the identity of amino acids incorporated into proteins during protein synthesis is almost universally conserved. Mitochondrial genomes feature deviations from the standard genetic code, including the reassignment of two arginine codons to stop codons. The protein required for translation termination at these noncanonical stop codons to release the newly synthesized polypeptides is not currently known. In this study, we used gene editing and ribosomal profiling in combination with cryo-electron microscopy to establish that mitochondrial release factor 1 (mtRF1) detects noncanonical stop codons in human mitochondria by a previously unknown mechanism of codon recognition. We discovered that binding of mtRF1 to the decoding center of the ribosome stabilizes a highly unusual conformation in the messenger RNA in which the ribosomal RNA participates in specific recognition of the noncanonical stop codons.
Asunto(s)
Codón de Terminación , Mitocondrias , Terminación de la Cadena Péptídica Traduccional , Factores de Terminación de Péptidos , Humanos , Microscopía por Crioelectrón , Mitocondrias/genética , Mitocondrias/metabolismo , Factores de Terminación de Péptidos/química , Conformación ProteicaRESUMEN
In all organisms, fatty acid synthesis is achieved in variations of a common cyclic reaction pathway by stepwise, iterative elongation of precursors with two-carbon extender units. In bacteria, all individual reaction steps are carried out by monofunctional dissociated enzymes, whereas in eukaryotes the fatty acid synthases (FASs) have evolved into large multifunctional enzymes that integrate the whole process of fatty acid synthesis. During the last few years, important advances in understanding the structural and functional organization of eukaryotic FASs have been made through a combination of biochemical, electron microscopic and X-ray crystallographic approaches. They have revealed the strikingly different architectures of the two distinct types of eukaryotic FASs, the fungal and the animal enzyme system. Fungal FAS is a 2·6 MDa α6ß6 heterododecamer with a barrel shape enclosing two large chambers, each containing three sets of active sites separated by a central wheel-like structure. It represents a highly specialized micro-compartment strictly optimized for the production of saturated fatty acids. In contrast, the animal FAS is a 540 kDa X-shaped homodimer with two lateral reaction clefts characterized by a modular domain architecture and large extent of conformational flexibility that appears to contribute to catalytic efficiency.
Asunto(s)
Eucariontes/enzimología , Ácido Graso Sintasas/química , Ácido Graso Sintasas/metabolismo , Animales , Dominio Catalítico , Cristalografía por Rayos X , Hongos/enzimología , HumanosRESUMEN
Pupylation is the post-translational modification of lysine side chains with prokaryotic ubiquitin-like protein (Pup) that targets proteins for proteasomal degradation in mycobacteria and other members of Actinobacteria. Pup ligase PafA and depupylase Dop are the two enzymes acting in this pathway. Although they share close structural and sequence homology indicative of a common evolutionary origin, they catalyze opposing reactions. Here, we report a series of high-resolution crystal structures of Dop in different functional states along the reaction pathway, including Pup-bound states in distinct conformations. In combination with biochemical analysis, the structures explain the role of the C-terminal residue of Pup in ATP hydrolysis, the process that generates the catalytic phosphate in the active site, and suggest a role for the Dop-loop as an allosteric sensor for Pup-binding and ATP cleavage.
Asunto(s)
Amidohidrolasas/química , Proteínas Bacterianas/química , Fosfatos/química , Ubiquitinas/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Amidohidrolasas/metabolismo , Proteínas Bacterianas/metabolismo , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Enlace de Hidrógeno , Hidrólisis , Modelos Moleculares , Fosfatos/metabolismo , Conformación Proteica , Procesamiento Proteico-Postraduccional , Ubiquitinas/metabolismoRESUMEN
Mitochondrial ribosomes are specialized for the synthesis of membrane proteins responsible for oxidative phosphorylation. Mammalian mitoribosomes have diverged considerably from the ancestral bacterial ribosomes and feature dramatically reduced ribosomal RNAs. The structural basis of the mammalian mitochondrial ribosome assembly is currently not well understood. Here we present eight distinct assembly intermediates of the human large mitoribosomal subunit involving seven assembly factors. We discover that the NSUN4-MTERF4 dimer plays a critical role in the process by stabilizing the 16S rRNA in a conformation that exposes the functionally important regions of rRNA for modification by the MRM2 methyltransferase and quality control interactions with the conserved mitochondrial GTPase MTG2 that contacts the sarcin-ricin loop and the immature active site. The successive action of these factors leads to the formation of the peptidyl transferase active site of the mitoribosome and the folding of the surrounding rRNA regions responsible for interactions with tRNAs and the small ribosomal subunit.
Asunto(s)
Ribosomas Mitocondriales/química , Peptidil Transferasas/química , Dominio Catalítico , Microscopía por Crioelectrón , Humanos , Metiltransferasas/química , Metiltransferasas/metabolismo , Ribosomas Mitocondriales/metabolismo , Modelos Moleculares , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/metabolismo , Conformación de Ácido Nucleico , Peptidil Transferasas/metabolismo , Multimerización de Proteína , ARN Ribosómico/química , ARN Ribosómico/metabolismo , Subunidades Ribosómicas Grandes/química , Subunidades Ribosómicas Grandes/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Programmed ribosomal frameshifting is a key event during translation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome that allows synthesis of the viral RNA-dependent RNA polymerase and downstream proteins. Here, we present the cryo-electron microscopy structure of a translating mammalian ribosome primed for frameshifting on the viral RNA. The viral RNA adopts a pseudoknot structure that lodges at the entry to the ribosomal messenger RNA (mRNA) channel to generate tension in the mRNA and promote frameshifting, whereas the nascent viral polyprotein forms distinct interactions with the ribosomal tunnel. Biochemical experiments validate the structural observations and reveal mechanistic and regulatory features that influence frameshifting efficiency. Finally, we compare compounds previously shown to reduce frameshifting with respect to their ability to inhibit SARS-CoV-2 replication, establishing coronavirus frameshifting as a target for antiviral intervention.