RESUMEN
The formation of new ribosomes is tightly coordinated with cell growth and proliferation. In eukaryotes, the correct assembly of all ribosomal proteins and RNAs follows an intricate scheme of maturation and rearrangement steps across three cellular compartments: the nucleolus, nucleoplasm, and cytoplasm. We demonstrate that usnic acid, a lichen secondary metabolite, inhibits the maturation of the large ribosomal subunit in yeast. We combine biochemical characterization of pre-ribosomal particles with a quantitative single-particle cryo-EM approach to monitor changes in nucleolar particle populations upon drug treatment. Usnic acid rapidly blocks the transition from nucleolar state B to C of Nsa1-associated pre-ribosomes, depleting key maturation factors such as Dbp10 and hindering pre-rRNA processing. This primary nucleolar block rapidly rebounds on earlier stages of the pathway which highlights the regulatory linkages between different steps. In summary, we provide an in-depth characterization of the effect of usnic acid on ribosome biogenesis, which may have implications for its reported anti-cancer activities.
Asunto(s)
Benzofuranos , Nucléolo Celular , Microscopía por Crioelectrón , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Benzofuranos/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Nucléolo Celular/metabolismo , Nucléolo Celular/efectos de los fármacos , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Ribosomas/efectos de los fármacos , ARN Ribosómico/metabolismo , Subunidades Ribosómicas Grandes/metabolismo , Precursores del ARN/metabolismo , Precursores del ARN/genética , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Líquenes/metabolismoRESUMEN
Ribosomal RNA modifications are introduced by specific enzymes during ribosome assembly in bacteria. Deletion of individual modification enzymes has a minor effect on bacterial growth, ribosome biogenesis, and translation, which has complicated the definition of the function of the enzymes and their products. We have constructed an Escherichia coli strain lacking 10 genes encoding enzymes that modify 23S rRNA around the peptidyl-transferase center. This strain exhibits severely compromised growth and ribosome assembly, especially at lower temperatures. Re-introduction of the individual modification enzymes allows for the definition of their functions. The results demonstrate that in addition to previously known RlmE, also RlmB, RlmKL, RlmN and RluC facilitate large ribosome subunit assembly. RlmB and RlmKL have functions in ribosome assembly independent of their modification activities. While the assembly stage specificity of rRNA modification enzymes is well established, this study demonstrates that there is a mutual interdependence between the rRNA modification process and large ribosome subunit assembly.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , ARN Ribosómico , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Metiltransferasas/metabolismo , Metiltransferasas/genética , Subunidades Ribosómicas Grandes/metabolismo , Subunidades Ribosómicas Grandes/genética , Subunidades Ribosómicas Grandes Bacterianas/metabolismo , Subunidades Ribosómicas Grandes Bacterianas/genética , Ribosomas/metabolismo , Ribosomas/genética , ARN Ribosómico/metabolismo , ARN Ribosómico/genética , ARN Ribosómico 23S/metabolismo , ARN Ribosómico 23S/genética , ARN Ribosómico 23S/químicaRESUMEN
BACKGROUND: NOL12 5'-3' exoribonucleases, conserved among eukaryotes, play important roles in pre-rRNA processing, ribosome assembly and export. The most well-described yeast counterpart, Rrp17, is required for maturation of 5.8 and 25S rRNAs, whereas human hNOL12 is crucial for the separation of the large (LSU) and small (SSU) ribosome subunit rRNA precursors. RESULTS: In this study we demonstrate that plant AtNOL12 is also involved in rRNA biogenesis, specifically in the processing of the LSU rRNA precursor, 27S pre-rRNA. Importantly, the absence of AtNOL12 alters the expression of many ribosomal protein and ribosome biogenesis genes. These changes could potentially exacerbate rRNA biogenesis defects, or, conversely, they might stem from the disturbed ribosome assembly caused by delayed pre-rRNA processing. Moreover, exposure of the nol12 mutant to stress factors, including heat and pathogen Pseudomonas syringae, enhances the observed molecular phenotypes, linking pre-rRNA processing to stress response pathways. The aberrant rRNA processing, dependent on AtNOL12, could impact ribosome function, as suggested by improved mutant resistance to ribosome-targeting antibiotics. CONCLUSION: Despite extensive studies, the pre-rRNA processing pathway in plants remains insufficiently characterized. Our investigation reveals the involvement of AtNOL12 in the maturation of rRNA precursors, correlating this process to stress response in Arabidopsis. These findings contribute to a more comprehensive understanding of plant ribosome biogenesis.
Asunto(s)
Arabidopsis , Humanos , Arabidopsis/genética , Arabidopsis/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Ribosómico/genética , Ribosomas/genética , Ribosomas/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Procesamiento Postranscripcional del ARN , Subunidades Ribosómicas Grandes/metabolismo , Plantas/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Large ribosomal subunit precursors (pre-LSUs) are primarily synthesized in the nucleolus. At an undetermined step in their assembly, they are released into the nucleoplasm. Structural models of yeast pre-LSUs at various stages of assembly have been collected using cryo-EM. However, which cryo-EM model is closest to the final nucleolar intermediate of the LSU has yet to be determined. To elucidate the mechanisms of the release of pre-LSUs from the nucleolus, we assayed effects of depleting or knocking out two yeast ribosome biogenesis factors (RiBi factors), Puf6 and Nog2, and two ribosomal proteins, uL2 and eL43. These proteins function during or stabilize onto pre-LSUs between the late nucleolar stages to early nucleoplasmic stages of ribosome biogenesis. By characterizing the phenotype of these four mutants, we determined that a particle that is intermediate between the cryo-EM model State NE1 and State NE2 likely represents the final nucleolar assembly intermediate of the LSU. We conclude that the release of the RiBi factors Nip7, Nop2 and Spb1 and the subsequent stabilization of rRNA domains IV and V may be key triggers for the release of pre-LSUs from the nucleolus.
Asunto(s)
Proteínas Ribosómicas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Grandes/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , ARN Ribosómico/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Ribosome biogenesis is one of the biggest consumers of cellular energy. More than 20 genetic diseases (ribosomopathies) and multiple cancers arise from defects in the production of the 40S (SSU) and 60S (LSU) ribosomal subunits. Defects in the production of either the SSU or LSU result in p53 induction through the accumulation of the 5S RNP, an LSU assembly intermediate. While the mechanism is understood for the LSU, it is still unclear how SSU production defects induce p53 through the 5S RNP since the production of the two subunits is believed to be uncoupled. Here, we examined the response to SSU production defects to understand how this leads to the activation of p53 via the 5S RNP. We found that p53 activation occurs rapidly after SSU production is blocked, prior to changes in mature ribosomal RNA (rRNA) levels but correlated with early, middle and late SSU pre-rRNA processing defects. Furthermore, both nucleolar/nuclear LSU maturation, in particular late stages in 5.8S rRNA processing, and pre-LSU export were affected by SSU production defects. We have therefore uncovered a novel connection between the SSU and LSU production pathways in human cells, which explains how p53 is induced in response to SSU production defects.
Asunto(s)
Subunidades Ribosómicas Grandes , Subunidades Ribosómicas Pequeñas , Proteína p53 Supresora de Tumor , Humanos , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Grandes/metabolismo , Subunidades Ribosómicas Pequeñas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
During transcription of eukaryotic ribosomal DNA in the nucleolus, assembly checkpoints exist that guarantee the formation of stable precursors of small and large ribosomal subunits. While the formation of an early large subunit assembly checkpoint precedes the separation of small and large subunit maturation, its mechanism of action and function remain unknown. Here, we report the cryo-electron microscopy structure of the yeast co-transcriptional large ribosomal subunit assembly intermediate that serves as a checkpoint. The structure provides the mechanistic basis for how quality-control pathways are established through co-transcriptional ribosome assembly factors, that structurally interrogate, remodel and, together with ribosomal proteins, cooperatively stabilize correctly folded pre-ribosomal RNA. Our findings thus provide a molecular explanation for quality control during eukaryotic ribosome assembly in the nucleolus.
Asunto(s)
ARN Ribosómico , Proteínas de Saccharomyces cerevisiae , Microscopía por Crioelectrón , ARN Ribosómico/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Grandes/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismoRESUMEN
Understanding the assembly principles of biological macromolecular complexes remains a significant challenge, due to the complexity of the systems and the difficulties in developing experimental approaches. As a ribonucleoprotein complex, the ribosome serves as a model system for the profiling of macromolecular complex assembly. In this work, we report an ensemble of large ribosomal subunit intermediate structures that accumulate during synthesis in a near-physiological and co-transcriptional in vitro reconstitution system. Thirteen pre-50S intermediate maps covering the entire assembly process were resolved using cryo-EM single-particle analysis and heterogeneous subclassification. Segmentation of the set of density maps reveals that the 50S ribosome intermediates assemble based on fourteen cooperative assembly blocks, including the smallest assembly core reported to date, which is composed of a 600-nucleotide-long folded rRNA and three ribosomal proteins. The cooperative blocks assemble onto the assembly core following defined dependencies, revealing the parallel pathways at both early and late assembly stages of the 50S subunit.
Asunto(s)
ARN Ribosómico , Ribosomas , Ribosomas/genética , Ribosomas/metabolismo , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Grandes/metabolismoRESUMEN
Many cellular processes, including ribosome biogenesis, are regulated through post-transcriptional RNA modifications. Here, a genome-wide analysis of the human mitochondrial transcriptome shows that 2'-O-methylation is limited to residues of the mitoribosomal large subunit (mtLSU) 16S mt-rRNA, introduced by MRM1, MRM2 and MRM3, with the modifications installed by the latter two proteins being interdependent. MRM2 controls mitochondrial respiration by regulating mitoribosome biogenesis. In its absence, mtLSU particles (visualized by cryo-EM at the resolution of 2.6 Å) present disordered RNA domains, partial occupancy of bL36m and bound MALSU1:L0R8F8:mtACP anti-association module, allowing five mtLSU biogenesis intermediates with different intersubunit interface configurations to be placed along the assembly pathway. However, mitoribosome biogenesis does not depend on the methyltransferase activity of MRM2. Disruption of the MRM2 Drosophila melanogaster orthologue leads to mitochondria-related developmental arrest. This work identifies a key checkpoint during mtLSU assembly, essential to maintain mitochondrial homeostasis.
Asunto(s)
Proteínas de Drosophila/metabolismo , Metiltransferasas/metabolismo , Ribosomas Mitocondriales/metabolismo , Biosíntesis de Proteínas , Subunidades Ribosómicas Grandes/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Masculino , Metilación , Metiltransferasas/genética , ARN Ribosómico 16S/metabolismo , Proteínas Ribosómicas/metabolismoRESUMEN
In yeast and human cells many of the ribosomal proteins (r-proteins) are required for the stabilisation and productive processing of rRNA precursors. Functional coupling of r-protein assembly with the stabilisation and maturation of subunit precursors potentially promotes the production of ribosomes with defined composition. To further decipher mechanisms of such an intrinsic quality control pathway we analysed here the contribution of three yeast large ribosomal subunit r-proteins rpL2 (uL2), rpL25 (uL23) and rpL34 (eL34) for intermediate nuclear subunit folding steps. Structure models obtained from single particle cryo-electron microscopy analyses provided evidence for specific and hierarchic effects on the stable positioning and remodelling of large ribosomal subunit domains. Based on these structural and previous biochemical data we discuss possible mechanisms of r-protein dependent hierarchic domain arrangement and the resulting impact on the stability of misassembled subunits.
Asunto(s)
Proteínas Fúngicas/metabolismo , Precursores del ARN/metabolismo , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Pliegue de Proteína , Subunidades Ribosómicas Grandes/metabolismoRESUMEN
Assembly of the mitoribosome is largely enigmatic and involves numerous assembly factors. Little is known about their function and the architectural transitions of the pre-ribosomal intermediates. Here, we solve cryo-EM structures of the human 39S large subunit pre-ribosomes, representing five distinct late states. Besides the MALSU1 complex used as bait for affinity purification, we identify several assembly factors, including the DDX28 helicase, MRM3, GTPBP10 and the NSUN4-mTERF4 complex, all of which keep the 16S rRNA in immature conformations. The late transitions mainly involve rRNA domains IV and V, which form the central protuberance, the intersubunit side and the peptidyltransferase center of the 39S subunit. Unexpectedly, we find deacylated tRNA in the ribosomal E-site, suggesting a role in 39S assembly. Taken together, our study provides an architectural inventory of the distinct late assembly phase of the human 39S mitoribosome.
Asunto(s)
Ribosomas Mitocondriales/metabolismo , Subunidades Ribosómicas Grandes/metabolismo , Línea Celular , Codón sin Sentido/genética , Microscopía por Crioelectrón , ARN Helicasas DEAD-box , Humanos , Metiltransferasas/metabolismo , Ribosomas Mitocondriales/ultraestructura , Modelos Moleculares , Proteínas de Unión al GTP Monoméricas/metabolismo , ARN Ribosómico 16S/química , ARN Ribosómico 16S/ultraestructura , ARN de Transferencia/metabolismo , Subunidades Ribosómicas Grandes/ultraestructuraRESUMEN
Mitochondrial ribosomes (mitoribosomes) synthesize a critical set of proteins essential for oxidative phosphorylation. Therefore, mitoribosomal function is vital to the cellular energy supply. Mitoribosome biogenesis follows distinct molecular pathways that remain poorly understood. Here, we determine the cryo-EM structures of mitoribosomes isolated from human cell lines with either depleted or overexpressed mitoribosome assembly factor GTPBP5, allowing us to capture consecutive steps during mitoribosomal large subunit (mt-LSU) biogenesis. Our structures provide essential insights into the last steps of 16S rRNA folding, methylation and peptidyl transferase centre (PTC) completion, which require the coordinated action of nine assembly factors. We show that mammalian-specific MTERF4 contributes to the folding of 16S rRNA, allowing 16 S rRNA methylation by MRM2, while GTPBP5 and NSUN4 promote fine-tuning rRNA rearrangements leading to PTC formation. Moreover, our data reveal an unexpected involvement of the elongation factor mtEF-Tu in mt-LSU assembly, where mtEF-Tu interacts with GTPBP5, similar to its interaction with tRNA during translational elongation.
Asunto(s)
Ribosomas Mitocondriales/química , Subunidades Ribosómicas Grandes/química , Línea Celular , 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 , Complejos Multiproteicos , Factor Tu de Elongación Peptídica/química , Factor Tu de Elongación Peptídica/metabolismo , Peptidil Transferasas/química , Peptidil Transferasas/metabolismo , Unión Proteica , Pliegue del ARN , ARN Ribosómico 16S/química , ARN Ribosómico 16S/metabolismo , Subunidades Ribosómicas Grandes/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Ribosome biogenesis requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. Particularly, maturation of the peptidyl transferase center (PTC) is mediated by conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial large ribosomal subunit (mtLSU) using endogenous complex purification, in vitro reconstitution and cryo-EM. Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch and progression to a near-mature PTC state. Additionally, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results provide a framework for understanding step-wise PTC folding as a critical conserved quality control checkpoint.
Asunto(s)
Proteínas de Unión al GTP/química , Ribosomas Mitocondriales/química , Proteínas de Unión al GTP Monoméricas/química , Microscopía por Crioelectrón , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Humanos , Metiltransferasas/química , Metiltransferasas/metabolismo , Ribosomas Mitocondriales/metabolismo , Modelos Moleculares , Proteínas de Unión al GTP Monoméricas/metabolismo , Complejos Multiproteicos , Biogénesis de Organelos , Peptidil Transferasas/química , Peptidil Transferasas/metabolismo , Pliegue de Proteína , ARN Ribosómico/química , ARN Ribosómico/metabolismo , Subunidades Ribosómicas Grandes/química , Subunidades Ribosómicas Grandes/metabolismo , Factores de Transcripción/química , Factores de Transcripción/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
Alopecia, neurologic defects, and endocrinopathy (ANE) syndrome is a rare ribosomopathy known to be caused by a p.(Leu351Pro) variant in the essential, conserved, nucleolar large ribosomal subunit (60S) assembly factor RBM28. We report the second family of ANE syndrome to date and a female pediatric ANE syndrome patient. The patient presented with alopecia, craniofacial malformations, hypoplastic pituitary, and hair and skin abnormalities. Unlike the previously reported patients with the p.(Leu351Pro) RBM28 variant, this ANE syndrome patient possesses biallelic precursor messenger RNA (pre-mRNA) splicing variants at the 5' splice sites of exon 5 (ΔE5) and exon 8 (ΔE8) of RBM28 (NM_018077.2:c.[541+1_541+2delinsA]; [946G > T]). In silico analyses and minigene splicing experiments in cells indicate that each splice variant specifically causes skipping of its respective mutant exon. Because the ΔE5 variant results in an in-frame 31 amino acid deletion (p.(Asp150_Lys180del)) in RBM28 while the ΔE8 variant leads to a premature stop codon in exon 9, we predicted that the ΔE5 variant would produce partially functional RBM28 but the ΔE8 variant would not produce functional protein. Using a yeast model, we demonstrate that the ΔE5 variant does indeed lead to reduced overall growth and large subunit ribosomal RNA (rRNA) production and pre-rRNA processing. In contrast, the ΔE8 variant is comparably null, implying that the partially functional ΔE5 RBM28 protein enables survival but precludes correct development. This discovery further defines the underlying molecular pathology of ANE syndrome to include genetic variants that cause aberrant splicing in RBM28 pre-mRNA and highlights the centrality of nucleolar processes in human genetic disease.
Asunto(s)
Alopecia/metabolismo , Nucléolo Celular/metabolismo , Enfermedades del Sistema Endocrino/metabolismo , Discapacidad Intelectual/metabolismo , Empalme del ARN , Proteínas de Unión al ARN/metabolismo , Subunidades Ribosómicas Grandes/metabolismo , Adulto , Alopecia/genética , Brasil , Enfermedades del Sistema Endocrino/genética , Exones , Femenino , Células HEK293 , Cabello/metabolismo , Humanos , Lactante , Discapacidad Intelectual/genética , Masculino , Linaje , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Subunidades Ribosómicas Grandes/genética , Saccharomyces cerevisiae , Adulto JovenRESUMEN
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
In plant cells, chloroplast gene expression is predominantly controlled through post-transcriptional regulation. Such fine-tuning is vital for precisely orchestrating protein complex assembly as for the photosynthesis machinery and for quickly responding to environmental changes. While regulation of chloroplast protein synthesis is of central importance, little is known about the degree and nature of the regulatory network, mainly due to challenges associated with the specific isolation of transient ribosome interactors. Here, we established a ribosome affinity purification method, which enabled us to broadly uncover putative ribosome-associated proteins in chloroplasts. Endogenously tagging of a protein of the large or small subunit revealed not only interactors of the holo complex, but also preferential interactors of the two subunits. This includes known canonical regulatory proteins as well as several new proteins belonging to the categories of protein and RNA regulation, photosystem biogenesis, redox control and metabolism. The sensitivity of the here applied screen was validated for various transiently interacting proteins. We further provided evidence for the existence of a ribosome-associated Nα-acetyltransferase in chloroplasts and its ability to acetylate substrate proteins at their N-terminus. The broad set of ribosome interactors underscores the potential to regulate chloroplast gene expression on the level of protein synthesis.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Ribosomas/metabolismo , Espectrometría de Masas en Tándem/métodos , Acetilación , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Fraccionamiento Celular/métodos , Chlamydomonas reinhardtii/genética , Regulación de la Expresión Génica de las Plantas , Separación Inmunomagnética , Espectrometría de Masas , Modelos Moleculares , Acetiltransferasas N-Terminal/aislamiento & purificación , Acetiltransferasas N-Terminal/metabolismo , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Procesamiento Proteico-Postraduccional , Subunidades Ribosómicas Grandes/metabolismo , Subunidades Ribosómicas Pequeñas/metabolismoRESUMEN
In translation elongation, two translational guanosine triphosphatase (trGTPase) factors EF1A and EF2 alternately bind to the ribosome and promote polypeptide elongation. The ribosomal stalk is a multimeric ribosomal protein complex which plays an essential role in the recruitment of EF1A and EF2 to the ribosome and their GTP hydrolysis for efficient and accurate translation elongation. However, due to the flexible nature of the ribosomal stalk, its structural dynamics and mechanism of action remain unclear. Here, we applied high-speed atomic force microscopy (HS-AFM) to directly visualize the action of the archaeal ribosomal heptameric stalk complex, aP0â¢(aP1â¢aP1)3 (P-stalk). HS-AFM movies clearly demonstrated the wobbling motion of the P-stalk on the large ribosomal subunit where the stalk base adopted two conformational states, a predicted canonical state, and a newly identified flipped state. Moreover, we showed that up to seven molecules of archaeal EF1A (aEF1A) and archaeal EF2 (aEF2) assembled around the ribosomal P-stalk, corresponding to the copy number of the common C-terminal factor-binding site of the P-stalk. These results provide visual evidence for the factor-pooling mechanism by the P-stalk within the ribosome and reveal that the ribosomal P-stalk promotes translation elongation by increasing the local concentration of translational GTPase factors.
Asunto(s)
Proteínas Arqueales/química , Factores de Elongación Enlazados a GTP Fosfohidrolasas/metabolismo , Microscopía de Fuerza Atómica/métodos , Proteínas Ribosómicas/química , Subunidades Ribosómicas Grandes/química , Proteínas Arqueales/metabolismo , Escherichia coli/genética , Factores de Elongación Enlazados a GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Extensión de la Cadena Peptídica de Translación , Pyrococcus horikoshii/química , Pyrococcus horikoshii/genética , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Grandes/metabolismoRESUMEN
Enterococcus faecalis is a gram-positive organism responsible for serious infections in humans, but as with many bacterial pathogens, resistance has rendered a number of commonly used antibiotics ineffective. Here, we report the cryo-EM structure of the E. faecalis 70S ribosome to a global resolution of 2.8 Å. Structural differences are clustered in peripheral and solvent exposed regions when compared with Escherichia coli, whereas functional centres, including antibiotic binding sites, are similar to other bacterial ribosomes. Comparison of intersubunit conformations among five classes obtained after three-dimensional classification identifies several rotated states. Large ribosomal subunit protein bL31, which forms intersubunit bridges to the small ribosomal subunit, assumes different conformations in the five classes, revealing how contacts to the small subunit are maintained throughout intersubunit rotation. A tRNA observed in one of the five classes is positioned in a chimeric pe/E position in a rotated ribosomal state. The 70S ribosome structure of E. faecalis now extends our knowledge of bacterial ribosome structures and may serve as a basis for the development of novel antibiotic compounds effective against this pathogen.
Asunto(s)
Enterococcus faecalis/ultraestructura , Subunidades Ribosómicas Grandes/ultraestructura , Antibacterianos/metabolismo , Sitios de Unión , Microscopía por Crioelectrón , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , Conformación Proteica , Subunidades Ribosómicas Grandes/metabolismoRESUMEN
Recognition of a start codon by the initiator aminoacyl-tRNA determines the reading frame of messenger RNA (mRNA) translation by the ribosome. In eukaryotes, the GTPase eIF5B collaborates in the correct positioning of the initiator Met-tRNAiMet on the ribosome in the later stages of translation initiation, gating entrance into elongation. Leveraging the long residence time of eIF5B on the ribosome recently identified by single-molecule fluorescence measurements, we determine the cryoEM structure of the naturally long-lived ribosome complex with eIF5B and Met-tRNAiMet immediately before transition into elongation. The structure uncovers an unexpected, eukaryotic specific and dynamic fidelity checkpoint implemented by eIF5B in concert with components of the large ribosomal subunit.
Asunto(s)
Factores Eucarióticos de Iniciación/química , Factores Eucarióticos de Iniciación/metabolismo , Extensión de la Cadena Peptídica de Translación , Iniciación de la Cadena Peptídica Traduccional , Subunidades Ribosómicas Grandes/metabolismo , Acilación , Anticodón , Microscopía por Crioelectrón , Factores Eucarióticos de Iniciación/genética , Guanosina Difosfato/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , ARN de Transferencia de Metionina/química , ARN de Transferencia de Metionina/metabolismo , Subunidades Ribosómicas Grandes/química , Subunidades Ribosómicas Grandes/genética , Subunidades Ribosómicas Grandes de Eucariotas , Subunidades Ribosómicas Pequeñas de Eucariotas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/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.