RESUMO
Mitochondria are cellular organelles responsible for generation of chemical energy in the process called oxidative phosphorylation. They originate from a bacterial ancestor and maintain their own genome, which is expressed by designated, mitochondrial transcription and translation machineries that differ from those operating for nuclear gene expression. In particular, the mitochondrial protein synthesis machinery is structurally and functionally very different from that governing eukaryotic, cytosolic translation. Despite harbouring their own genetic information, mitochondria are far from being independent of the rest of the cell and, conversely, cellular fitness is closely linked to mitochondrial function. Mitochondria depend heavily on the import of nuclear-encoded proteins for gene expression and function, and hence engage in extensive inter-compartmental crosstalk to regulate their proteome. This connectivity allows mitochondria to adapt to changes in cellular conditions and also mediates responses to stress and mitochondrial dysfunction. With a focus on mammals and yeast, we review fundamental insights that have been made into the biogenesis, architecture and mechanisms of the mitochondrial translation apparatus in the past years owing to the emergence of numerous near-atomic structures and a considerable amount of biochemical work. Moreover, we discuss how cellular mitochondrial protein expression is regulated, including aspects of mRNA and tRNA maturation and stability, roles of auxiliary factors, such as translation regulators, that adapt mitochondrial translation rates, and the importance of inter-compartmental crosstalk with nuclear gene expression and cytosolic translation and how it enables integration of mitochondrial translation into the cellular context.
Assuntos
Eucariotos/genética , Mitocôndrias/genética , Biossíntese de Proteínas/genética , Transcrição Gênica , Animais , Regulação da Expressão Gênica/genética , Humanos , Fosforilação Oxidativa , RNA Mensageiro/genética , RNA de Transferência/genéticaRESUMO
Mitochondrial ribosomes (mitoribosomes) perform protein synthesis inside mitochondria, the organelles responsible for energy conversion and adenosine triphosphate production in eukaryotic cells. Throughout evolution, mitoribosomes have become functionally specialized for synthesizing mitochondrial membrane proteins, and this has been accompanied by large changes to their structure and composition. We review recent high-resolution structural data that have provided unprecedented insight into the structure and function of mitoribosomes in mammals and fungi.
Assuntos
DNA Mitocondrial/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Ribossomos Mitocondriais/ultraestrutura , Biossíntese de Proteínas , Subunidades Ribossômicas/ultraestrutura , Animais , Antibacterianos/farmacologia , Evolução Biológica , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , DNA Mitocondrial/metabolismo , Mamíferos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/química , Ribossomos Mitocondriais/metabolismo , Modelos Moleculares , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Subunidades Ribossômicas/química , Subunidades Ribossômicas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
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.
Assuntos
Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Chaetomium/metabolismo , Microscopia Crioeletrônica , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Humanos , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/ultraestrutura , Subunidades Ribossômicas Maiores de Eucariotos/ultraestrutura , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Alinhamento de SequênciaRESUMO
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.
Assuntos
COVID-19 , Quirópteros , Animais , Quirópteros/genética , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Domínios Proteicos , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Eukaryotic translation initiation requires the recruitment of the large, multiprotein eIF3 complex to the 40S ribosomal subunit. We present X-ray structures of all major components of the minimal, six-subunit Saccharomyces cerevisiae eIF3 core. These structures, together with electron microscopy reconstructions, cross-linking coupled to mass spectrometry, and integrative structure modeling, allowed us to position and orient all eIF3 components on the 40Sâ eIF1 complex, revealing an extended, modular arrangement of eIF3 subunits. Yeast eIF3 engages 40S in a clamp-like manner, fully encircling 40S to position key initiation factors on opposite ends of the mRNA channel, providing a platform for the recruitment, assembly, and regulation of the translation initiation machinery. The structures of eIF3 components reported here also have implications for understanding the architecture of the mammalian 43S preinitiation complex and the complex of eIF3, 40S, and the hepatitis C internal ribosomal entry site RNA.
Assuntos
Fator de Iniciação 1 em Eucariotos/química , Fator de Iniciação 3 em Eucariotos/química , Iniciação Traducional da Cadeia Peptídica , Subunidades Ribossômicas Menores de Eucariotos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Dimerização , Fator de Iniciação 1 em Eucariotos/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Hepacivirus/química , Humanos , Mamíferos/metabolismo , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Ribonucleoproteínas/química , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de SequênciaRESUMO
The mitochondrial translation system originates from a bacterial ancestor but has substantially diverged in the course of evolution. Here, we use single-particle cryo-electron microscopy (cryo-EM) as a screening tool to identify mitochondrial translation termination mechanisms and to describe them in molecular detail. We show how mitochondrial release factor 1a releases the nascent chain from the ribosome when it encounters the canonical stop codons UAA and UAG. Furthermore, we define how the peptidyl-tRNA hydrolase ICT1 acts as a rescue factor on mitoribosomes that have stalled on truncated messages to recover them for protein synthesis. Finally, we present structural models detailing the process of mitochondrial ribosome recycling to explain how a dedicated elongation factor, mitochondrial EFG2 (mtEFG2), has specialized for cooperation with the mitochondrial ribosome recycling factor to dissociate the mitoribosomal subunits at the end of the translation process.
Assuntos
Mitocôndrias/fisiologia , Ribossomos Mitocondriais/metabolismo , Terminação Traducional da Cadeia Peptídica/fisiologia , Animais , Hidrolases de Éster Carboxílico , Códon de Terminação , Microscopia Crioeletrônica/métodos , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Terminação Traducional da Cadeia Peptídica/genética , Fator G para Elongação de Peptídeos/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/fisiologia , Ribossomos/metabolismoRESUMO
Coronaviruses are a group of related RNA viruses that cause respiratory diseases in humans and animals. Understanding the mechanisms of translation regulation during coronaviral infections is critical for developing antiviral therapies and preventing viral spread. Translation of the viral single-stranded RNA genome in the host cell cytoplasm is an essential step in the life cycle of coronaviruses, which affects the cellular mRNA translation landscape in many ways. Here we discuss various viral strategies of translation control, including how members of the Betacoronavirus genus shut down host cell translation and suppress host innate immune functions, as well as the role of the viral non-structural protein 1 (Nsp1) in the process. We also outline the fate of viral RNA, considering stress response mechanisms triggered in infected cells, and describe how unique viral RNA features contribute to programmed ribosomal -1 frameshifting, RNA editing, and translation shutdown evasion.
Assuntos
Infecções por Coronavirus , Coronavirus , Animais , Humanos , Coronavirus/genética , Infecções por Coronavirus/genética , Betacoronavirus/fisiologia , Antivirais/farmacologia , RNA Viral/genéticaRESUMO
Ribosomes have been suggested to directly control gene regulation, but regulatory roles for ribosomal RNA (rRNA) remain largely unexplored. Expansion segments (ESs) consist of multitudes of tentacle-like rRNA structures extending from the core ribosome in eukaryotes. ESs are remarkably variable in sequence and size across eukaryotic evolution with largely unknown functions. In characterizing ribosome binding to a regulatory element within a Homeobox (Hox) 5' UTR, we identify a modular stem-loop within this element that binds to a single ES, ES9S. Engineering chimeric, "humanized" yeast ribosomes for ES9S reveals that an evolutionary change in the sequence of ES9S endows species-specific binding of Hoxa9 mRNA to the ribosome. Genome editing to site-specifically disrupt the Hoxa9-ES9S interaction demonstrates the functional importance for such selective mRNA-rRNA binding in translation control. Together, these studies unravel unexpected gene regulation directly mediated by rRNA and how ribosome evolution drives translation of critical developmental regulators.
Assuntos
Proteínas de Homeodomínio/genética , Biossíntese de Proteínas/genética , RNA Ribossômico/ultraestrutura , Ribossomos/genética , Regiões 5' não Traduzidas/genética , Regulação da Expressão Gênica/genética , Genes Homeobox/genética , Proteínas de Homeodomínio/ultraestrutura , Conformação de Ácido Nucleico , RNA Mensageiro/genética , RNA Ribossômico/genética , Ribossomos/ultraestrutura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Especificidade da EspécieRESUMO
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.
Assuntos
Ribossomos Mitocondriais/química , RNA Ribossômico/metabolismo , Subunidades Ribossômicas Maiores/química , Trypanosoma brucei brucei/metabolismo , Microscopia Crioeletrônica , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Ribossomos Mitocondriais/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Ribossômico/química , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores/metabolismo , Trypanosoma brucei brucei/genéticaRESUMO
Cotranslational processing of newly synthesized proteins is fundamental for correct protein maturation. Protein biogenesis factors are thought to bind nascent polypeptides not before they exit the ribosomal tunnel. Here, we identify a nascent chain recognition mechanism deep inside the ribosomal tunnel by an essential eukaryotic cytosolic chaperone. The nascent polypeptide-associated complex (NAC) inserts the N-terminal tail of its ß subunit (N-ßNAC) into the ribosomal tunnel to sense substrates directly upon synthesis close to the peptidyl-transferase center. N-ßNAC escorts the growing polypeptide to the cytosol and relocates to an alternate binding site on the ribosomal surface. Using C. elegans as an in vivo model, we demonstrate that the tunnel-probing activity of NAC is essential for organismal viability and critical to regulate endoplasmic reticulum (ER) protein transport by controlling ribosome-Sec61 translocon interactions. Thus, eukaryotic protein maturation relies on the early sampling of nascent chains inside the ribosomal tunnel.
Assuntos
Proteínas de Caenorhabditis elegans/biossíntese , Caenorhabditis elegans/metabolismo , Retículo Endoplasmático/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Canais de Translocação SEC/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Retículo Endoplasmático/genética , Humanos , Ribossomos/genética , Canais de Translocação SEC/genética , Saccharomyces cerevisiaeRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Proteínas Oncogênicas/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Microscopia Crioeletrônica , Cristalografia por Raios X , Fator de Iniciação 2 em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/genética , Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/genética , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Complexos Multiproteicos , Mutação , Conformação de Ácido Nucleico , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Ligação Proteica , Conformação Proteica , RNA Mensageiro/química , RNA Mensageiro/genética , RNA de Transferência/química , RNA de Transferência/genética , Subunidades Ribossômicas Menores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/genética , Relação Estrutura-Atividade , TransfecçãoRESUMO
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.
Assuntos
Proteínas Mitocondriais/química , Ribossomos Mitocondriais/química , Fosforilação Oxidativa , RNA Ribossômico/química , Proteínas Ribossômicas/química , Subunidades Ribossômicas/química , Linhagem Celular , Microscopia Crioeletrônica , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/metabolismo , Modelos Moleculares , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas/genética , Subunidades Ribossômicas/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMO
Mitochondria are eukaryotic organelles of bacterial origin where respiration takes place to produce cellular chemical energy. These reactions are catalyzed by the respiratory chain complexes located in the inner mitochondrial membrane. Notably, key components of the respiratory chain complexes are encoded on the mitochondrial chromosome and their expression relies on a dedicated mitochondrial translation machinery. Defects in the mitochondrial gene expression machinery lead to a variety of diseases in humans mostly affecting tissues with high energy demand such as the nervous system, the heart, or the muscles. The mitochondrial translation system has substantially diverged from its bacterial ancestor, including alterations in the mitoribosomal architecture, multiple changes to the set of translation factors and striking reductions in otherwise conserved tRNA elements. Although a number of structures of mitochondrial ribosomes from different species have been determined, our mechanistic understanding of the mitochondrial translation cycle remains largely unexplored. Here, we present two cryo-EM reconstructions of human mitochondrial elongation factor G1 bound to the mammalian mitochondrial ribosome at two different steps of the tRNA translocation reaction during translation elongation. Our structures explain the mechanism of tRNA and mRNA translocation on the mitoribosome, the regulation of mtEFG1 activity by the ribosomal GTPase-associated center, and the basis of decreased susceptibility of mtEFG1 to the commonly used antibiotic fusidic acid.
Assuntos
Proteínas Mitocondriais/química , Ribossomos Mitocondriais/química , Ribossomos Mitocondriais/ultraestrutura , Fator G para Elongação de Peptídeos/química , Biossíntese de Proteínas , RNA Mitocondrial/química , RNA de Transferência/química , Animais , Microscopia Crioeletrônica , Humanos , Membranas Mitocondriais/química , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/metabolismo , Fator G para Elongação de Peptídeos/genética , Fator G para Elongação de Peptídeos/metabolismo , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , SuínosRESUMO
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.
Assuntos
Microscopia Crioeletrônica , Mamíferos , Mitocôndrias/ultraestrutura , Iniciação Traducional da Cadeia Peptídica , Animais , Códon de Iniciação/genética , Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/ultraestrutura , Mitocôndrias/química , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/ultraestrutura , Modelos Moleculares , RNA Mitocondrial/química , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , RNA Mitocondrial/ultraestrutura , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo , RNA de Transferência de Metionina/ultraestruturaRESUMO
Biogenesis of ribosomal subunits involves enzymatic modifications of rRNA that fine-tune functionally important regions. The universally conserved prokaryotic dimethyltransferase KsgA sequentially modifies two universally conserved adenosine residues in helix 45 of the small ribosomal subunit rRNA, which is in proximity of the decoding site. Here we present the cryo-EM structure of Escherichia coli KsgA bound to an E. coli 30S at a resolution of 3.1 Å. The high-resolution structure reveals how KsgA recognizes immature rRNA and binds helix 45 in a conformation where one of the substrate nucleotides is flipped-out into the active site. We suggest that successive processing of two adjacent nucleotides involves base-flipping of the rRNA, which allows modification of the second substrate nucleotide without dissociation of the enzyme. Since KsgA is homologous to the essential eukaryotic methyltransferase Dim1 involved in 40S maturation, these results have also implications for understanding eukaryotic ribosome maturation.
Assuntos
Adenosina/metabolismo , Escherichia coli/enzimologia , Metiltransferases/química , Adenosina/química , Microscopia Crioeletrônica , Metiltransferases/metabolismo , Modelos Moleculares , Conformação Proteica , Subunidades Ribossômicas Menores de Bactérias/química , Especificidade por SubstratoRESUMO
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.
Assuntos
Microscopia Crioeletrônica , Simulação de Acoplamento Molecular , Proteínas Ribossômicas/ultraestrutura , Subunidades Ribossômicas Menores de Eucariotos/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae/ultraestrutura , Citoplasma , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/ultraestrutura , Conformação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/ultraestrutura , Dobramento de RNA , RNA Ribossômico/química , RNA Ribossômico/ultraestrutura , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/ultraestrutura , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/isolamento & purificação , Subunidades Ribossômicas Menores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificaçãoRESUMO
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.
Assuntos
Cloroplastos , Ribossomos/química , Ribossomos/ultraestrutura , Spinacia oleracea , Microscopia Crioeletrônica , Modelos Moleculares , RNA Ribossômico/química , RNA Ribossômico/ultraestrutura , Proteínas Ribossômicas/química , Proteínas Ribossômicas/ultraestruturaRESUMO
The universally conserved signal recognition particle (SRP) system mediates the targeting of membrane proteins to the translocon in a multistep process controlled by GTP hydrolysis. Here we present the 2.6 Å crystal structure of the GTPase domains of the E. coli SRP protein (Ffh) and its receptor (FtsY) in complex with the tetraloop and the distal region of SRP-RNA, trapped in the activated state in presence of GDP:AlF4. The structure reveals the atomic details of FtsY recruitment and, together with biochemical experiments, pinpoints G83 as the key RNA residue that stimulates GTP hydrolysis. Insertion of G83 into the FtsY active site orients a single glutamate residue provided by Ffh (E277), triggering GTP hydrolysis and complex disassembly at the end of the targeting cycle. The complete conservation of the key residues of the SRP-RNA and the SRP protein implies that the suggested chemical mechanism of GTPase activation is applicable across all kingdoms.
Assuntos
Proteínas de Bactérias/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , RNA/genética , Receptores Citoplasmáticos e Nucleares/genética , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/metabolismo , Compostos de Alumínio/farmacologia , Proteínas de Bactérias/metabolismo , Sequência de Bases , Domínio Catalítico/efeitos dos fármacos , Domínio Catalítico/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fluoretos/farmacologia , Ativadores de GTP Fosfo-Hidrolase/farmacologia , Guanosina Difosfato/genética , Guanosina Difosfato/metabolismo , Hidrólise/efeitos dos fármacos , Dados de Sequência Molecular , RNA/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Análise de Sequência de DNARESUMO
[This corrects the article DOI: 10.1371/journal.pbio.1002557.].