RESUMEN
Researchers commonly anneal metals, alloys, and semiconductors to repair defects and improve microstructures via recrystallization. Theoretical studies indicate that simulated annealing on biological macromolecules helps predict the final structures with minimum free energy. Experimental validation of this homogenizing effect and further exploration of its applications are fascinating scientific questions that remain elusive. Here, we chose the apo-state 70S ribosome from Escherichia coli as a model, wherein the 30S subunit undergoes a thermally driven intersubunit rotation and exhibits substantial structural flexibility as well as distinct free energy. We experimentally demonstrate that annealing at a fast cooling rate enhances the 70S ribosome homogeneity and improves local resolution on the 30S subunit. After annealing, the 70S ribosome is in a nonrotated state with respect to corresponding intermediate structures in unannealed or heated ribosomes. Manifold-based analysis further indicates that the annealed 70S ribosome takes a narrow conformational distribution and exhibits a minimum-energy state in the free-energy landscape. Our experimental results offer a facile yet robust approach to enhance protein stability, which is ideal for high-resolution cryogenic electron microscopy. Beyond structure determination, annealing shows great potential for synchronizing proteins on a single-molecule level and can be extended to study protein folding and explore conformational and energy landscapes.
Asunto(s)
Conformación Proteica , Proteínas Ribosómicas/ultraestructura , Ribosomas/fisiología , Microscopía por Crioelectrón , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , ARN Ribosómico/metabolismo , ARN Ribosómico/ultraestructura , Proteínas Ribosómicas/metabolismo , Ribosomas/ultraestructuraRESUMEN
Sequence variation in a widespread, recurrent, structured RNA 3D motif, the Sarcin/Ricin (S/R), was studied to address three related questions: First, how do the stabilities of structured RNA 3D motifs, composed of non-Watson-Crick (non-WC) basepairs, compare to WC-paired helices of similar length and sequence? Second, what are the effects on the stabilities of such motifs of isosteric and non-isosteric base substitutions in the non-WC pairs? And third, is there selection for particular base combinations in non-WC basepairs, depending on the temperature regime to which an organism adapts? A survey of large and small subunit rRNAs from organisms adapted to different temperatures revealed the presence of systematic sequence variations at many non-WC paired sites of S/R motifs. UV melting analysis and enzymatic digestion assays of oligonucleotides containing the motif suggest that more stable motifs tend to be more rigid. We further found that the base substitutions at non-Watson-Crick pairing sites can significantly affect the thermodynamic stabilities of S/R motifs and these effects are highly context specific indicating the importance of base-stacking and base-phosphate interactions on motif stability. This study highlights the significance of non-canonical base pairs and their contributions to modulating the stability and flexibility of RNA molecules.
Asunto(s)
Motivos de Nucleótidos/genética , ARN Ribosómico/ultraestructura , ARN/ultraestructura , Emparejamiento Base/genética , Cristalografía por Rayos X , Enlace de Hidrógeno/efectos de los fármacos , Conformación de Ácido Nucleico/efectos de los fármacos , ARN/efectos de los fármacos , ARN/genética , ARN Ribosómico/efectos de los fármacos , ARN Ribosómico/genética , Ricina/farmacologíaRESUMEN
Adenosine-to-inosine (A-to-I) editing of eukaryotic cellular RNAs is essential for protection against auto-immune disorders. Editing is carried out by ADAR1, whose innate immune response-specific cytoplasmic isoform possesses a Z-DNA binding domain (Zα) of unknown function. Zα also binds to CpG repeats in RNA, which are a hallmark of Z-RNA formation. Unexpectedly, Zα has been predicted - and in some cases even shown - to bind to specific regions within mRNA and rRNA devoid of such repeats. Here, we use NMR, circular dichroism, and other biophysical approaches to demonstrate and characterize the binding of Zα to mRNA and rRNA fragments. Our results reveal a broad range of RNA sequences that bind to Zα and adopt Z-RNA conformations. Binding is accompanied by destabilization of neighboring A-form regions which is similar in character to what has been observed for B-Z-DNA junctions. The binding of Zα to non-CpG sequences is specific, cooperative and occurs with an affinity in the low micromolar range. This work allows us to propose a model for how Zα could influence the RNA binding specificity of ADAR1.
Asunto(s)
Adenosina Desaminasa/metabolismo , Elementos Alu/genética , Dominios Proteicos , ARN Ribosómico/metabolismo , Proteínas de Unión al ARN/metabolismo , Adenosina Desaminasa/genética , Adenosina Desaminasa/aislamiento & purificación , Adenosina Desaminasa/ultraestructura , Dicroismo Circular , Inmunidad Innata , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , Motivo de Reconocimiento de ARN , ARN Ribosómico/genética , ARN Ribosómico/inmunología , ARN Ribosómico/ultraestructura , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas de Unión al ARN/ultraestructura , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructuraRESUMEN
Many antibiotics inhibit bacterial growth by binding to the ribosome and interfering with protein biosynthesis. Macrolides represent one of the most successful classes of ribosome-targeting antibiotics. The main clinically relevant mechanism of resistance to macrolides is dimethylation of the 23S rRNA nucleotide A2058, located in the drug-binding site, a reaction catalyzed by Erm-type rRNA methyltransferases. Here, we present the crystal structure of the Erm-dimethylated 70S ribosome at 2.4 Å resolution, together with the structures of unmethylated 70S ribosome functional complexes alone or in combination with macrolides. Altogether, our structural data do not support previous models and, instead, suggest a principally new explanation of how A2058 dimethylation confers resistance to macrolides. Moreover, high-resolution structures of two macrolide antibiotics bound to the unmodified ribosome reveal a previously unknown role of the desosamine moiety in drug binding, laying a foundation for the rational knowledge-based design of macrolides that can overcome Erm-mediated resistance.
Asunto(s)
Macrólidos/metabolismo , ARN Ribosómico/ultraestructura , Ribosomas/ultraestructura , Antibacterianos/farmacología , Farmacorresistencia Bacteriana/genética , Macrólidos/farmacología , Metilación , ARN Ribosómico/genética , ARN Ribosómico 23S/genética , ARN Ribosómico 23S/metabolismo , ARN Ribosómico 23S/ultraestructura , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Homeodominio/genética , Biosíntesis de Proteínas/genética , ARN Ribosómico/ultraestructura , Ribosomas/genética , Regiones no Traducidas 5'/genética , Regulación de la Expresión Génica/genética , Genes Homeobox/genética , Proteínas de Homeodominio/ultraestructura , Conformación de Ácido Nucleico , ARN Mensajero/genética , ARN Ribosómico/genética , Ribosomas/ultraestructura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Especificidad de la EspecieRESUMEN
Bacterial ribosomal RNAs are synthesized by a dedicated, conserved transcription-elongation complex that transcribes at high rates, shields RNA polymerase from premature termination, and supports co-transcriptional RNA folding, modification, processing, and ribosomal subunit assembly by presently unknown mechanisms. We have determined cryo-electron microscopy structures of complete Escherichia coli ribosomal RNA transcription elongation complexes, comprising RNA polymerase; DNA; RNA bearing an N-utilization-site-like anti-termination element; Nus factors A, B, E, and G; inositol mono-phosphatase SuhB; and ribosomal protein S4. Our structures and structure-informed functional analyses show that fast transcription and anti-termination involve suppression of NusA-stabilized pausing, enhancement of NusG-mediated anti-backtracking, sequestration of the NusG C-terminal domain from termination factor ρ, and the ρ blockade. Strikingly, the factors form a composite RNA chaperone around the RNA polymerase RNA-exit tunnel, which supports co-transcriptional RNA folding and annealing of distal RNA regions. Our work reveals a polymerase/chaperone machine required for biosynthesis of functional ribosomes.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/genética , Chaperonas Moleculares/genética , Proteínas Ribosómicas/genética , Ribosomas/genética , Sitios de Unión/genética , Microscopía por Crioelectrón , Escherichia coli/genética , Escherichia coli/ultraestructura , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestructura , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/ultraestructura , Biosíntesis de Proteínas/genética , Pliegue del ARN/genética , ARN Ribosómico/genética , ARN Ribosómico/ultraestructura , Proteínas Ribosómicas/ultraestructura , Ribosomas/ultraestructura , Factores de Elongación Transcripcional/química , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/ultraestructuraRESUMEN
Ribonucleic acids (RNAs) play essential roles in living cells. Many of them fold into defined three-dimensional (3D) structures to perform functions. Recent advances in single-particle cryo-electron microscopy (cryo-EM) have enabled structure determinations of RNA to atomic resolutions. However, most RNA molecules are structurally flexible, limiting the resolution of their structures solved by cryo-EM. In modeling these molecules, several computational methods are limited by the requirement of massive computational resources and/or the low efficiency in exploring large-scale structural variations. Here we use hierarchical natural move Monte Carlo (HNMMC), which takes advantage of collective motions for groups of nucleic acid residues, to refine RNA structures into their cryo-EM maps, preserving atomic details in the models. After validating the method on a simulated density map of tRNA, we applied it to objectively obtain the model of the folding intermediate for the specificity domain of ribonuclease P from Bacillus subtilis and refine a flexible ribosomal RNA (rRNA) expansion segment from the Mycobacterium tuberculosis (Mtb) ribosome in different conformational states. Finally, we used HNMMC to model atomic details and flexibility for two distinct conformations of the complete genomic RNA (gRNA) inside MS2, a single-stranded RNA virus, revealing multiple pathways for its capsid assembly.
Asunto(s)
Método de Montecarlo , Virus ARN/ultraestructura , ARN Ribosómico/ultraestructura , ARN de Transferencia/ultraestructura , ARN/ultraestructura , Ribosomas/ultraestructura , Bacillus subtilis/enzimología , Proteínas de la Cápside/genética , Proteínas de la Cápside/ultraestructura , Modelos Moleculares , ARN/genética , Virus ARN/genética , ARN Ribosómico/genética , ARN de Transferencia/genética , Ribonucleasa P/genética , Ribonucleasa P/ultraestructura , Ribosomas/genéticaRESUMEN
The vast majority of eukaryotic cells contain mitochondria, essential powerhouses and metabolic hubs1. These organelles have a bacterial origin and were acquired during an early endosymbiosis event2. Mitochondria possess specialized gene expression systems composed of various molecular machines, including the mitochondrial ribosomes (mitoribosomes). Mitoribosomes are in charge of translating the few essential mRNAs still encoded by mitochondrial genomes3. While chloroplast ribosomes strongly resemble those of bacteria4,5, mitoribosomes have diverged significantly during evolution and present strikingly different structures across eukaryotic species6-10. In contrast to animals and trypanosomatids, plant mitoribosomes have unusually expanded ribosomal RNAs and have conserved the short 5S rRNA, which is usually missing in mitoribosomes11. We have previously characterized the composition of the plant mitoribosome6, revealing a dozen plant-specific proteins in addition to the common conserved mitoribosomal proteins. In spite of the tremendous recent advances in the field, plant mitoribosomes remained elusive to high-resolution structural investigations and the plant-specific ribosomal features of unknown structures. Here, we present a cryo-electron microscopy study of the plant 78S mitoribosome from cauliflower at near-atomic resolution. We show that most of the plant-specific ribosomal proteins are pentatricopeptide repeat proteins (PPRs) that deeply interact with the plant-specific rRNA expansion segments. These additional rRNA segments and proteins reshape the overall structure of the plant mitochondrial ribosome, and we discuss their involvement in the membrane association and mRNA recruitment prior to translation initiation. Finally, our structure unveils an rRNA-constructive phase of mitoribosome evolution across eukaryotes.
Asunto(s)
Brassica/ultraestructura , Ribosomas Mitocondriales/ultraestructura , ARN de Planta/ultraestructura , ARN Ribosómico/ultraestructura , Brassica/genética , Microscopía por Crioelectrón , Evolución Molecular , Modelos Moleculares , Proteínas de Plantas/ultraestructura , Proteínas Ribosómicas/ultraestructuraRESUMEN
Bacteria harbor a number GTPases that function in the assembly of the ribosome and are essential for growth. RbgA is one of these GTPases and is required for the assembly of the 50S subunit in most bacteria. Homologs of this protein are also implicated in the assembly of the large subunit of the mitochondrial and eukaryotic ribosome. We present here the cryo-electron microscopy structure of RbgA bound to a Bacillus subtilis 50S subunit assembly intermediate (45SRbgA particle) that accumulates in cells upon RbgA depletion. Binding of RbgA at the P site of the immature particle stabilizes functionally important rRNA helices in the A and P-sites, prior to the completion of the maturation process of the subunit. The structure also reveals the location of the highly conserved N-terminal end of RbgA containing the catalytic residue Histidine 9. The derived model supports a mechanism of GTP hydrolysis, and it shows that upon interaction of RbgA with the 45SRbgA particle, Histidine 9 positions itself near the nucleotide potentially acting as the catalytic residue with minimal rearrangements. This structure represents the first visualization of the conformational changes induced by an assembly factor in a bacterial subunit intermediate.
Asunto(s)
GTP Fosfohidrolasas/química , ARN Ribosómico/química , Proteínas Ribosómicas/química , Bacillus subtilis/química , Bacillus subtilis/genética , Microscopía por Crioelectrón , GTP Fosfohidrolasas/ultraestructura , Hidrólisis , Modelos Moleculares , Conformación Proteica , ARN Ribosómico/genética , ARN Ribosómico/ultraestructura , Proteínas Ribosómicas/ultraestructura , Subunidades Ribosómicas Grandes Bacterianas/química , Subunidades Ribosómicas Grandes Bacterianas/genética , Subunidades Ribosómicas Grandes Bacterianas/ultraestructura , Ribosomas/genética , Ribosomas/ultraestructuraRESUMEN
Mitochondrial ribosomes (mitoribosomes) are large ribonucleoprotein complexes that synthesize proteins encoded by the mitochondrial genome. An extensive cellular machinery responsible for ribosome assembly has been described only for eukaryotic cytosolic ribosomes. Here we report that the assembly of the small mitoribosomal subunit in Trypanosoma brucei involves a large number of factors and proceeds through the formation of assembly intermediates, which we analyzed by using cryo-electron microscopy. One of them is a 4-megadalton complex, referred to as the small subunit assemblosome, in which we identified 34 factors that interact with immature ribosomal RNA (rRNA) and recognize its functionally important regions. The assembly proceeds through large-scale conformational changes in rRNA coupled with successive incorporation of mitoribosomal proteins, providing an example for the complexity of the ribosomal assembly process in mitochondria.
Asunto(s)
Proteínas Mitocondriales/ultraestructura , Ribosomas Mitocondriales/ultraestructura , ARN Ribosómico/ultraestructura , Proteínas Ribosómicas/ultraestructura , Trypanosoma brucei brucei/química , Microscopía por Crioelectrón , Modelos Moleculares , Conformación de Ácido Nucleico , Estructura Cuaternaria de Proteína , Interferencia de ARN , Estabilidad del ARNRESUMEN
Fibrillar centers (FCs) and dense fibrillar components (DFCs) are essential morphologically distinct sub-regions of mammalian cell nucleoli for rDNA transcription and pre-rRNA processing. Here, we report that a human nucleolus consists of several dozen FC/DFC units, each containing 2-3 transcriptionally active rDNAs at the FC/DFC border. Pre-rRNA processing factors, such as fibrillarin (FBL), form 18-24 clusters that further assemble into the DFC surrounding the FC. Mechanistically, the 5' end of nascent 47S pre-rRNA binds co-transcriptionally to the RNA-binding domain of FBL. FBL diffuses to the DFC, where local self-association via its glycine- and arginine-rich (GAR) domain forms phase-separated clusters to immobilize FBL-interacting pre-rRNA, thus promoting directional traffic of nascent pre-rRNA while facilitating pre-rRNA processing and DFC formation. These results unveil FC/DFC ultrastructures in nucleoli and suggest a conceptual framework for considering nascent RNA sorting using multivalent interactions of their binding proteins.
Asunto(s)
Nucléolo Celular/metabolismo , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Ribosómico/metabolismo , Transporte Activo de Núcleo Celular , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Nucléolo Celular/genética , Nucléolo Celular/ultraestructura , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Femenino , Células HEK293 , Células HeLa , Humanos , Conformación de Ácido Nucleico , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Precursores del ARN/genética , Precursores del ARN/ultraestructura , ARN Ribosómico/genética , ARN Ribosómico/ultraestructuraRESUMEN
Rix7 is an essential type II AAA-ATPase required for the formation of the large ribosomal subunit. Rix7 has been proposed to utilize the power of ATP hydrolysis to drive the removal of assembly factors from pre-60S particles, but the mechanism of release is unknown. Rix7's mammalian homolog, NVL2 has been linked to cancer and mental illness disorders, highlighting the need to understand the molecular mechanisms of this essential machine. Here we report the cryo-EM reconstruction of the tandem AAA domains of Rix7 which form an asymmetric stacked homohexameric ring. We trapped Rix7 with a polypeptide in the central channel, revealing Rix7's role as a molecular unfoldase. The structure establishes that type II AAA-ATPases lacking the aromatic-hydrophobic motif within the first AAA domain can engage a substrate throughout the entire central channel. The structure also reveals that Rix7 contains unique post-α7 insertions within both AAA domains important for Rix7 function.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/ultraestructura , Microscopía por Crioelectrón/métodos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/ultraestructura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructura , ATPasas Asociadas con Actividades Celulares Diversas/ultraestructura , ARN Ribosómico/metabolismo , ARN Ribosómico/ultraestructura , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Ribosomal RNA (rRNA) plays key functional and architectural roles in ribosomes. Using electron microscopy, we determined the atomic structure of a highly divergent ribosome found in mitochondria of Trypanosoma brucei, a unicellular parasite that causes sleeping sickness in humans. The trypanosomal mitoribosome features the smallest rRNAs and contains more proteins than all known ribosomes. The structure shows how the proteins have taken over the role of architectural scaffold from the rRNA: They form an autonomous outer shell that surrounds the entire particle and stabilizes and positions the functionally important regions of the rRNA. Our results also reveal the "minimal" set of conserved rRNA and protein components shared by all ribosomes that help us define the most essential functional elements.
Asunto(s)
Evolución Molecular , Ribosomas Mitocondriales/química , Proteínas Protozoarias/química , Proteínas Ribosómicas/química , Trypanosoma brucei brucei/ultraestructura , Ribosomas Mitocondriales/ultraestructura , Modelos Moleculares , Proteínas Protozoarias/ultraestructura , ARN Ribosómico/química , ARN Ribosómico/ultraestructura , Proteínas Ribosómicas/ultraestructuraRESUMEN
Ribosome biogenesis involves numerous preribosomal RNA (pre-rRNA) processing events to remove internal and external transcribed spacer sequences, ultimately yielding three mature rRNAs. Removal of the internal transcribed spacer 2 spacer RNA is the final step in large subunit pre-rRNA processing and begins with endonucleolytic cleavage at the C2 site of 27SB pre-rRNA. C2 cleavage requires the hierarchical recruitment of 11 ribosomal proteins and 14 ribosome assembly factors. However, the function of these proteins in C2 cleavage remained unclear. In this study, we have performed a detailed analysis of the effects of depleting proteins required for C2 cleavage and interpreted these results using cryo-electron microscopy structures of assembling 60S subunits. This work revealed that these proteins are required for remodeling of several neighborhoods, including two major functional centers of the 60S subunit, suggesting that these remodeling events form a checkpoint leading to C2 cleavage. Interestingly, when C2 cleavage is directly blocked by depleting or inactivating the C2 endonuclease, assembly progresses through all other subsequent steps.
Asunto(s)
Precursores del ARN/ultraestructura , ARN Ribosómico/ultraestructura , Proteínas Ribosómicas/ultraestructura , Ribosomas/ultraestructura , Microscopía por Crioelectrón , Cristalografía por Rayos X , ADN Espaciador Ribosómico/genética , ADN Espaciador Ribosómico/ultraestructura , Precursores del ARN/química , Precursores del ARN/genética , ARN Ribosómico/química , ARN Ribosómico/genética , Proteínas Ribosómicas/biosíntesis , Proteínas Ribosómicas/genética , Ribosomas/química , Ribosomas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructuraRESUMEN
Early co-transcriptional events during eukaryotic ribosome assembly result in the formation of precursors of the small (40S) and large (60S) ribosomal subunits. A multitude of transient assembly factors regulate and chaperone the systematic folding of pre-ribosomal RNA subdomains. However, owing to a lack of structural information, the role of these factors during early nucleolar 60S assembly is not fully understood. Here we report cryo-electron microscopy (cryo-EM) reconstructions of the nucleolar pre-60S ribosomal subunit in different conformational states at resolutions of up to 3.4 Å. These reconstructions reveal how steric hindrance and molecular mimicry are used to prevent both premature folding states and binding of later factors. This is accomplished by the concerted activity of 21 ribosome assembly factors that stabilize and remodel pre-ribosomal RNA and ribosomal proteins. Among these factors, three Brix-domain proteins and their binding partners form a ring-like structure at ribosomal RNA (rRNA) domain boundaries to support the architecture of the maturing particle. The existence of mutually exclusive conformations of these pre-60S particles suggests that the formation of the polypeptide exit tunnel is achieved through different folding pathways during subsequent stages of ribosome assembly. These structures rationalize previous genetic and biochemical data and highlight the mechanisms that drive eukaryotic ribosome assembly in a unidirectional manner.
Asunto(s)
Nucléolo Celular/química , Microscopía por Crioelectrón , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/ultraestructura , Saccharomyces cerevisiae , Reactivos de Enlaces Cruzados/química , Modelos Moleculares , Imitación Molecular , Dominios Proteicos , Estabilidad Proteica , Pliegue del ARN , ARN Ribosómico/química , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN Ribosómico/ultraestructura , Reproducibilidad de los Resultados , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/ultraestructura , Subunidades Ribosómicas Grandes de Eucariotas/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructuraRESUMEN
The RNA exosome complex processes and degrades a wide range of transcripts, including ribosomal RNAs (rRNAs). We used cryo-electron microscopy to visualize the yeast nuclear exosome holocomplex captured on a precursor large ribosomal subunit (pre-60S) during 7S-to-5.8S rRNA processing. The cofactors of the nuclear exosome are sandwiched between the ribonuclease core complex (Exo-10) and the remodeled "foot" structure of the pre-60S particle, which harbors the 5.8S rRNA precursor. The exosome-associated helicase Mtr4 recognizes the preribosomal substrate by docking to specific sites on the 25S rRNA, captures the 3' extension of the 5.8S rRNA, and channels it toward Exo-10. The structure elucidates how the exosome forms a structural and functional unit together with its massive pre-60S substrate to process rRNA during ribosome maturation.
Asunto(s)
Complejo Multienzimático de Ribonucleasas del Exosoma/química , Exosomas/química , Ribosomas/química , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Núcleo Celular/química , Núcleo Celular/ultraestructura , Microscopía por Crioelectrón , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/ultraestructura , Complejo Multienzimático de Ribonucleasas del Exosoma/ultraestructura , Exosomas/ultraestructura , Conformación Proteica , Precursores del ARN/química , Precursores del ARN/ultraestructura , ARN Ribosómico/química , ARN Ribosómico/ultraestructura , ARN Ribosómico 5.8S/química , ARN Ribosómico 5.8S/ultraestructura , Ribosomas/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/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
Bacterial ribosomal protein S1 is the largest and highly flexible protein of the 30S subunit, and one of a few core ribosomal proteins for which a complete structure is lacking. S1 is thought to participate in transcription and translation. Best understood is the role of S1 in facilitating translation of mRNAs with structured 5' UTRs. Here, we present cryo-EM analyses of the 70S ribosome that reveal multiple conformations of S1. Based on comparison of several 3D maximum likelihood classification approaches in Frealign, we propose a streamlined strategy for visualizing a highly dynamic component of a large macromolecular assembly that itself exhibits high compositional and conformational heterogeneity. The resulting maps show how S1 docks at the ribosomal protein S2 near the mRNA exit channel. The globular OB-fold domains sample a wide area around the mRNA exit channel and interact with mobile tails of proteins S6 and S18. S1 also interacts with the mRNA entrance channel, where an OB-fold domain can be localized near S3 and S5. Our analyses suggest that S1 cooperates with other ribosomal proteins to form a dynamic mesh near the mRNA exit and entrance channels to modulate the binding, folding and movement of mRNA.
Asunto(s)
Microscopía por Crioelectrón/métodos , ARN Ribosómico/ultraestructura , Proteínas Ribosómicas/ultraestructura , Subunidades Ribosómicas Grandes/ultraestructura , Citosol/ultraestructura , Escherichia coli/genética , Escherichia coli/ultraestructura , Conformación Proteica , Proteínas Ribosómicas/química , Subunidades Ribosómicas Grandes/químicaRESUMEN
Chemical modifications of human ribosomal RNA (rRNA) are introduced during biogenesis and have been implicated in the dysregulation of protein synthesis, as is found in cancer and other diseases. However, their role in this phenomenon is unknown. Here we visualize more than 130 individual rRNA modifications in the three-dimensional structure of the human ribosome, explaining their structural and functional roles. In addition to a small number of universally conserved sites, we identify many eukaryote- or human-specific modifications and unique sites that form an extended shell in comparison to bacterial ribosomes, and which stabilize the RNA. Several of the modifications are associated with the binding sites of three ribosome-targeting antibiotics, or are associated with degenerate states in cancer, such as keto alkylations on nucleotide bases reminiscent of specialized ribosomes. This high-resolution structure of the human 80S ribosome paves the way towards understanding the role of epigenetic rRNA modifications in human diseases and suggests new possibilities for designing selective inhibitors and therapeutic drugs.