ABSTRACT
During the last ten years, developments in cryo-electron microscopy have transformed our understanding of eukaryotic ribosome assembly. As a result, the field has advanced from a list of the vast array of ribosome assembly factors toward an emerging molecular movie in which individual frames are represented by structures of stable ribosome assembly intermediates with complementary biochemical and genetic data. In this review, we discuss the mechanisms driving the assembly of yeast and human small and large ribosomal subunits. A particular emphasis is placed on the most recent findings that illustrate key concepts of ribosome assembly, such as folding of preribosomal RNA, the enforced chronology of assembly, enzyme-mediated irreversible transitions, and proofreading of preribosomal particles.
Subject(s)
Cryoelectron Microscopy , Ribosomal Proteins , Ribosomes , Humans , Ribosomes/metabolism , Ribosomes/ultrastructure , Ribosomes/chemistry , Ribosomes/genetics , Ribosomal Proteins/metabolism , Ribosomal Proteins/chemistry , Ribosomal Proteins/genetics , RNA, Ribosomal/metabolism , RNA, Ribosomal/chemistry , RNA, Ribosomal/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/genetics , Models, Molecular , Eukaryotic Cells/metabolism , Eukaryotic Cells/ultrastructure , RNA Folding , Ribosome Subunits, Small, Eukaryotic/metabolism , Ribosome Subunits, Small, Eukaryotic/chemistry , Ribosome Subunits, Small, Eukaryotic/genetics , Ribosome Subunits, Small, Eukaryotic/ultrastructure , AnimalsABSTRACT
All cells fold their genomes, including bacterial cells, where the chromosome is compacted into a domain-organized meshwork called the nucleoid. How compaction and domain organization arise is not fully understood. Here, we describe a method to estimate the average mesh size of the nucleoid in Escherichia coli. Using nucleoid mesh size and DNA concentration estimates, we find that the cytoplasm behaves as a poor solvent for the chromosome when the cell is considered as a simple semidilute polymer solution. Monte Carlo simulations suggest that a poor solvent leads to chromosome compaction and DNA density heterogeneity (i.e., domain formation) at physiological DNA concentration. Fluorescence microscopy reveals that the heterogeneous DNA density negatively correlates with ribosome density within the nucleoid, consistent with cryoelectron tomography data. Drug experiments, together with past observations, suggest the hypothesis that RNAs contribute to the poor solvent effects, connecting chromosome compaction and domain formation to transcription and intracellular organization.
Subject(s)
Chromosomes, Bacterial/chemistry , Escherichia coli/metabolism , Nucleic Acid Conformation , Solvents/chemistry , Transcription, Genetic , Aminoglycosides/pharmacology , Computer Simulation , DNA, Bacterial/chemistry , Diffusion , Escherichia coli/drug effects , Green Fluorescent Proteins/metabolism , Particle Size , RNA, Bacterial/metabolism , Ribosomes/metabolism , Ribosomes/ultrastructure , Transcription, Genetic/drug effectsABSTRACT
Manipulation of individual molecules with optical tweezers provides a powerful means of interrogating the structure and folding of proteins. Mechanical force is not only a relevant quantity in cellular protein folding and function, but also a convenient parameter for biophysical folding studies. Optical tweezers offer precise control in the force range relevant for protein folding and unfolding, from which single-molecule kinetic and thermodynamic information about these processes can be extracted. In this review, we describe both physical principles and practical aspects of optical tweezers measurements and discuss recent advances in the use of this technique for the study of protein folding. In particular, we describe the characterization of folding energy landscapes at high resolution, studies of structurally complex multidomain proteins, folding in the presence of chaperones, and the ability to investigate real-time cotranslational folding of a polypeptide.
Subject(s)
Escherichia coli/genetics , Molecular Chaperones/genetics , Optical Tweezers , Protein Biosynthesis , Proteome/chemistry , Ribosomes/genetics , Escherichia coli/metabolism , Humans , Kinetics , Microscopy, Atomic Force , Models, Molecular , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , Protein Binding , Protein Folding , Protein Interaction Domains and Motifs , Proteome/biosynthesis , Proteome/genetics , Proteostasis/genetics , Ribosomes/metabolism , Ribosomes/ultrastructure , ThermodynamicsABSTRACT
Folding of polypeptides begins during their synthesis on ribosomes. This process has evolved as a means for the cell to maintain proteostasis, by mitigating the risk of protein misfolding and aggregation. The capacity to now depict this cellular feat at increasingly higher resolution is providing insight into the mechanistic determinants that promote successful folding. Emerging from these studies is the intimate interplay between protein translation and folding, and within this the ribosome particle is the key player. Its unique structural properties provide a specialized scaffold against which nascent polypeptides can begin to form structure in a highly coordinated, co-translational manner. Here, we examine how, as a macromolecular machine, the ribosome modulates the intrinsic dynamic properties of emerging nascent polypeptide chains and guides them toward their biologically active structures.
Subject(s)
Escherichia coli/genetics , Molecular Chaperones/genetics , Protein Biosynthesis , Proteome/chemistry , Ribosomes/genetics , Cryoelectron Microscopy , Escherichia coli/metabolism , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , Protein Binding , Protein Folding , Protein Interaction Domains and Motifs , Proteome/biosynthesis , Proteome/genetics , Proteostasis/genetics , Proteostasis Deficiencies/genetics , Proteostasis Deficiencies/metabolism , Proteostasis Deficiencies/pathology , Ribosomes/metabolism , Ribosomes/ultrastructureABSTRACT
Stalled protein synthesis produces defective nascent chains that can harm cells. In response, cells degrade these nascent chains via a process called ribosome-associated quality control (RQC). Here, we review the irregularities in the translation process that cause ribosomes to stall as well as how cells use RQC to detect stalled ribosomes, ubiquitylate their tethered nascent chains, and deliver the ubiquitylated nascent chains to the proteasome. We additionally summarize how cells respond to RQC failure.
Subject(s)
Escherichia coli/genetics , Proteasome Endopeptidase Complex/metabolism , Protein Biosynthesis , Protein Processing, Post-Translational , Ribosomes/genetics , Escherichia coli/metabolism , Humans , Models, Molecular , Poly A/chemistry , Poly A/genetics , Poly A/metabolism , Proteasome Endopeptidase Complex/genetics , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Proteolysis , RNA Splicing , RNA Stability , Ribosomes/metabolism , Ribosomes/ultrastructure , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
The enteric nervous system (ENS) coordinates diverse functions in the intestine but has eluded comprehensive molecular characterization because of the rarity and diversity of cells. Here we develop two methods to profile the ENS of adult mice and humans at single-cell resolution: RAISIN RNA-seq for profiling intact nuclei with ribosome-bound mRNA and MIRACL-seq for label-free enrichment of rare cell types by droplet-based profiling. The 1,187,535 nuclei in our mouse atlas include 5,068 neurons from the ileum and colon, revealing extraordinary neuron diversity. We highlight circadian expression changes in enteric neurons, show that disease-related genes are dysregulated with aging, and identify differences between the ileum and proximal/distal colon. In humans, we profile 436,202 nuclei, recovering 1,445 neurons, and identify conserved and species-specific transcriptional programs and putative neuro-epithelial, neuro-stromal, and neuro-immune interactions. The human ENS expresses risk genes for neuropathic, inflammatory, and extra-intestinal diseases, suggesting neuronal contributions to disease.
Subject(s)
Enteric Nervous System/cytology , Enteric Nervous System/metabolism , Gene Expression Regulation, Developmental/genetics , Neurons/metabolism , Nissl Bodies/metabolism , RNA, Messenger/metabolism , Single-Cell Analysis/methods , Aging/genetics , Aging/metabolism , Animals , Circadian Clocks/genetics , Colon/cytology , Colon/metabolism , Endoplasmic Reticulum, Rough/genetics , Endoplasmic Reticulum, Rough/metabolism , Endoplasmic Reticulum, Rough/ultrastructure , Epithelial Cells/metabolism , Female , Genetic Predisposition to Disease/genetics , Humans , Ileum/cytology , Ileum/metabolism , Inflammation/genetics , Inflammation/metabolism , Intestinal Diseases/genetics , Intestinal Diseases/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Electron, Transmission , Nervous System Diseases/genetics , Nervous System Diseases/metabolism , Neuroglia/cytology , Neuroglia/metabolism , Neurons/cytology , Nissl Bodies/genetics , Nissl Bodies/ultrastructure , RNA, Messenger/genetics , RNA-Seq , Ribosomes/metabolism , Ribosomes/ultrastructure , Stromal Cells/metabolismABSTRACT
Multidrug resistance is a global threat as the clinically available potent antibiotic drugs are becoming exceedingly scarce. For example, increasing drug resistance among gram-positive bacteria is responsible for approximately one-third of nosocomial infections. As ribosomes are a major target for these drugs, they may serve as suitable objects for novel development of next-generation antibiotics. Three-dimensional structures of ribosomal particles from Staphylococcus aureus obtained by X-ray crystallography have shed light on fine details of drug binding sites and have revealed unique structural motifs specific for this pathogenic strain, which may be used for the design of novel degradable pathogen-specific, and hence, environmentally friendly drugs.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Bacterial Proteins/chemistry , Drug Design , Ribosomes/drug effects , Staphylococcus aureus/drug effects , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Cross Infection/drug therapy , Cross Infection/microbiology , Crystallography, X-Ray , Deinococcus/drug effects , Deinococcus/genetics , Deinococcus/metabolism , Drug Resistance, Multiple, Bacterial , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Models, Molecular , Ribosomes/metabolism , Ribosomes/ultrastructure , Staphylococcal Infections/drug therapy , Staphylococcal Infections/microbiology , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolism , Thermus thermophilus/drug effects , Thermus thermophilus/genetics , Thermus thermophilus/metabolismABSTRACT
In eukaryotes, accurate protein synthesis relies on a family of translational GTPases that pair with specific decoding factors to decipher the mRNA code on ribosomes. We present structures of the mammalian ribosome engaged with decoding factorâ GTPase complexes representing intermediates of translation elongation (aminoacyl-tRNAâ eEF1A), termination (eRF1â eRF3), and ribosome rescue (Pelotaâ Hbs1l). Comparative analyses reveal that each decoding factor exploits the plasticity of the ribosomal decoding center to differentially remodel ribosomal proteins and rRNA. This leads to varying degrees of large-scale ribosome movements and implies distinct mechanisms for communicating information from the decoding center to each GTPase. Additional structural snapshots of the translation termination pathway reveal the conformational changes that choreograph the accommodation of decoding factors into the peptidyl transferase center. Our results provide a structural framework for how different states of the mammalian ribosome are selectively recognized by the appropriate decoding factorâ GTPase complex to ensure translational fidelity.
Subject(s)
Protein Biosynthesis , RNA, Messenger/chemistry , Ribosomes/chemistry , Animals , Cryoelectron Microscopy , Endonucleases , Humans , Microfilament Proteins/metabolism , Models, Chemical , Models, Molecular , Nuclear Proteins , Peptide Elongation Factors/metabolism , Ribosomes/ultrastructureABSTRACT
The 90S pre-ribosome is an early biogenesis intermediate formed during co-transcriptional ribosome formation, composed of â¼70 assembly factors and several small nucleolar RNAs (snoRNAs) that associate with nascent pre-rRNA. We report the cryo-EM structure of the Chaetomium thermophilum 90S pre-ribosome, revealing how a network of biogenesis factors including 19 ß-propellers and large α-solenoid proteins engulfs the pre-rRNA. Within the 90S pre-ribosome, we identify the UTP-A, UTP-B, Mpp10-Imp3-Imp4, Bms1-Rcl1, and U3 snoRNP modules, which are organized around 5'-ETS and partially folded 18S rRNA. The U3 snoRNP is strategically positioned at the center of the 90S particle to perform its multiple tasks during pre-rRNA folding and processing. The architecture of the elusive 90S pre-ribosome gives unprecedented structural insight into the early steps of pre-rRNA maturation. Nascent rRNA that is co-transcriptionally folded and given a particular shape by encapsulation within a dedicated mold-like structure is reminiscent of how polypeptides use chaperone chambers for their protein folding.
Subject(s)
Chaetomium/chemistry , Organelle Biogenesis , Ribosomes/chemistry , Saccharomyces cerevisiae/chemistry , Chaetomium/classification , Cryoelectron Microscopy , Models, Molecular , RNA, Ribosomal, 18S/chemistry , Ribosome Subunits, Large, Eukaryotic/chemistry , Ribosome Subunits, Small, Eukaryotic/chemistry , Ribosomes/ultrastructureABSTRACT
About 20 years ago, the first three-dimensional (3D) reconstructions at subnanometer (<10-Å) resolution of an icosahedral virus assembly were obtained by cryogenic electron microscopy (cryo-EM) and single-particle analysis. Since then, thousands of structures have been determined to resolutions ranging from 30 Å to near atomic (<4 Å). Almost overnight, the recent development of direct electron detectors and the attendant improvement in analysis software have advanced the technology considerably. Near-atomic-resolution reconstructions can now be obtained, not only for megadalton macromolecular complexes or highly symmetrical assemblies but also for proteins of only a few hundred kilodaltons. We discuss the developments that led to this breakthrough in high-resolution structure determination by cryo-EM and point to challenges that lie ahead.
Subject(s)
Cryoelectron Microscopy/methods , Cryoelectron Microscopy/instrumentation , Eukaryotic Cells/ultrastructure , Macromolecular Substances/ultrastructure , Models, Molecular , Ribosomes/ultrastructure , SoftwareABSTRACT
Macromolecular machines, such as the ribosome, undergo large-scale conformational changes during their functional cycles. Although their mode of action is often compared to that of mechanical machines, a crucial difference is that, at the molecular dimension, thermodynamic effects dominate functional cycles, with proteins fluctuating stochastically between functional states defined by energetic minima on an energy landscape. Here, we have used cryo-electron microscopy to image ex-vivo-derived human polysomes as a source of actively translating ribosomes. Multiparticle refinement and 3D variability analysis allowed us to visualize a variety of native translation intermediates. Significantly populated states include not only elongation cycle intermediates in pre- and post-translocational states, but also eEF1A-containing decoding and termination/recycling complexes. Focusing on the post-translocational state, we extended this assessment to the single-residue level, uncovering striking details of ribosome-ligand interactions and identifying both static and functionally important dynamic elements.
Subject(s)
Protein Biosynthesis , Ribosomes/chemistry , Ribosomes/ultrastructure , Amino Acid Sequence , Cryoelectron Microscopy , Humans , Models, Molecular , Molecular Sequence Data , Phylogeny , RNA, Transfer/chemistry , Sequence Alignment , ThermodynamicsABSTRACT
Angiogenin, an RNase-A-family protein, promotes angiogenesis and has been implicated in cancer, neurodegenerative diseases and epigenetic inheritance1-10. After activation during cellular stress, angiogenin cleaves tRNAs at the anticodon loop, resulting in translation repression11-15. However, the catalytic activity of isolated angiogenin is very low, and the mechanisms of the enzyme activation and tRNA specificity have remained a puzzle3,16-23. Here we identify these mechanisms using biochemical assays and cryogenic electron microscopy (cryo-EM). Our study reveals that the cytosolic ribosome is the activator of angiogenin. A cryo-EM structure features angiogenin bound in the A site of the 80S ribosome. The C-terminal tail of angiogenin is rearranged by interactions with the ribosome to activate the RNase catalytic centre, making the enzyme several orders of magnitude more efficient in tRNA cleavage. Additional 80S-angiogenin structures capture how tRNA substrate is directed by the ribosome into angiogenin's active site, demonstrating that the ribosome acts as the specificity factor. Our findings therefore suggest that angiogenin is activated by ribosomes with a vacant A site, the abundance of which increases during cellular stress24-27. These results may facilitate the development of therapeutics to treat cancer and neurodegenerative diseases.
Subject(s)
Cryoelectron Microscopy , Ribonuclease, Pancreatic , Ribosomes , Humans , Anticodon/chemistry , Anticodon/genetics , Anticodon/metabolism , Anticodon/ultrastructure , Catalytic Domain , Cytosol/metabolism , Enzyme Activation , Models, Molecular , Ribonuclease, Pancreatic/chemistry , Ribonuclease, Pancreatic/metabolism , Ribonuclease, Pancreatic/ultrastructure , Ribosomes/metabolism , Ribosomes/chemistry , Ribosomes/ultrastructure , RNA Cleavage , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Substrate Specificity , Binding Sites , Stress, PhysiologicalABSTRACT
One of the most critical steps of protein synthesis is coupled translocation of messenger RNA (mRNA) and transfer RNAs (tRNAs) required to advance the mRNA reading frame by one codon. In eukaryotes, translocation is accelerated and its fidelity is maintained by elongation factor 2 (eEF2)1,2. At present, only a few snapshots of eukaryotic ribosome translocation have been reported3-5. Here we report ten high-resolution cryogenic-electron microscopy (cryo-EM) structures of the elongating eukaryotic ribosome bound to the full translocation module consisting of mRNA, peptidyl-tRNA and deacylated tRNA, seven of which also contained ribosome-bound, naturally modified eEF2. This study recapitulates mRNA-tRNA2-growing peptide module progression through the ribosome, from the earliest states of eEF2 translocase accommodation until the very late stages of the process, and shows an intricate network of interactions preventing the slippage of the translational reading frame. We demonstrate how the accuracy of eukaryotic translocation relies on eukaryote-specific elements of the 80S ribosome, eEF2 and tRNAs. Our findings shed light on the mechanism of translation arrest by the anti-fungal eEF2-binding inhibitor, sordarin. We also propose that the sterically constrained environment imposed by diphthamide, a conserved eukaryotic posttranslational modification in eEF2, not only stabilizes correct Watson-Crick codon-anticodon interactions but may also uncover erroneous peptidyl-tRNA, and therefore contribute to higher accuracy of protein synthesis in eukaryotes.
Subject(s)
Eukaryotic Cells , Protein Biosynthesis , RNA, Messenger , Reading Frames , Ribosomes , Anticodon/genetics , Anticodon/metabolism , Codon/genetics , Codon/metabolism , Cryoelectron Microscopy , Eukaryotic Cells/chemistry , Eukaryotic Cells/metabolism , Eukaryotic Cells/ultrastructure , Peptide Elongation Factor 2/antagonists & inhibitors , Peptide Elongation Factor 2/metabolism , Reading Frames/genetics , Ribosomes/chemistry , Ribosomes/metabolism , Ribosomes/ultrastructure , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Transfer/chemistry , RNA, Transfer/genetics , RNA, Transfer/metabolismABSTRACT
To conserve energy during starvation and stress, many organisms use hibernation factor proteins to inhibit protein synthesis and protect their ribosomes from damage1,2. In bacteria, two families of hibernation factors have been described, but the low conservation of these proteins and the huge diversity of species, habitats and environmental stressors have confounded their discovery3-6. Here, by combining cryogenic electron microscopy, genetics and biochemistry, we identify Balon, a new hibernation factor in the cold-adapted bacterium Psychrobacter urativorans. We show that Balon is a distant homologue of the archaeo-eukaryotic translation factor aeRF1 and is found in 20% of representative bacteria. During cold shock or stationary phase, Balon occupies the ribosomal A site in both vacant and actively translating ribosomes in complex with EF-Tu, highlighting an unexpected role for EF-Tu in the cellular stress response. Unlike typical A-site substrates, Balon binds to ribosomes in an mRNA-independent manner, initiating a new mode of ribosome hibernation that can commence while ribosomes are still engaged in protein synthesis. Our work suggests that Balon-EF-Tu-regulated ribosome hibernation is a ubiquitous bacterial stress-response mechanism, and we demonstrate that putative Balon homologues in Mycobacteria bind to ribosomes in a similar fashion. This finding calls for a revision of the current model of ribosome hibernation inferred from common model organisms and holds numerous implications for how we understand and study ribosome hibernation.
Subject(s)
Bacterial Proteins , Cold-Shock Response , Peptide Termination Factors , Protein Biosynthesis , Psychrobacter , Ribosomal Proteins , Ribosomes , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/ultrastructure , Peptide Elongation Factor Tu/chemistry , Peptide Elongation Factor Tu/metabolism , Peptide Elongation Factor Tu/ultrastructure , Ribosomal Proteins/chemistry , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Ribosomal Proteins/ultrastructure , Ribosomes/chemistry , Ribosomes/metabolism , Ribosomes/ultrastructure , Psychrobacter/chemistry , Psychrobacter/genetics , Psychrobacter/metabolism , Psychrobacter/ultrastructure , Cryoelectron Microscopy , Peptide Termination Factors/chemistry , Peptide Termination Factors/genetics , Peptide Termination Factors/metabolism , Peptide Termination Factors/ultrastructureABSTRACT
The cricket paralysis virus internal ribosome entry site (CrPV-IRES) is a folded structure in a viral mRNA that allows initiation of translation in the absence of any host initiation factors. By using recent advances in single-particle electron cryomicroscopy, we have solved the structure of CrPV-IRES bound to the ribosome of the yeast Kluyveromyces lactis in both the canonical and rotated states at overall resolutions of 3.7 and 3.8 Å, respectively. In both states, the pseudoknot PKI of the CrPV-IRES mimics a tRNA/mRNA interaction in the decoding center of the A site of the 40S ribosomal subunit. The structure and accompanying factor-binding data show that CrPV-IRES binding mimics a pretranslocation rather than initiation state of the ribosome. Translocation of the IRES by elongation factor 2 (eEF2) is required to bring the first codon of the mRNA into the A site and to allow the start of translation.
Subject(s)
Dicistroviridae/chemistry , Kluyveromyces/chemistry , Peptide Chain Initiation, Translational , RNA, Messenger/chemistry , RNA, Viral/chemistry , Ribosomes/chemistry , Cryoelectron Microscopy , Dicistroviridae/genetics , Kluyveromyces/metabolism , Models, Molecular , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Messenger/ultrastructure , RNA, Transfer/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , RNA, Viral/ultrastructure , Ribosomes/metabolism , Ribosomes/ultrastructureABSTRACT
Ribosome assembly is catalyzed by numerous trans-acting factors and coupled with irreversible pre-rRNA processing, driving the pathway toward mature ribosomal subunits. One decisive step early in this progression is removal of the 5' external transcribed spacer (5'-ETS), an RNA extension at the 18S rRNA that is integrated into the huge 90S pre-ribosome structure. Upon endo-nucleolytic cleavage at an internal site, A1, the 5'-ETS is separated from the 18S rRNA and degraded. Here we present biochemical and cryo-electron microscopy analyses that depict the RNA exosome, a major 3'-5' exoribonuclease complex, in a super-complex with the 90S pre-ribosome. The exosome is docked to the 90S through its co-factor Mtr4 helicase, a processive RNA duplex-dismantling helicase, which strategically positions the exosome at the base of 5'-ETS helices H9-H9', which are dislodged in our 90S-exosome structures. These findings suggest a direct role of the exosome in structural remodeling of the 90S pre-ribosome to drive eukaryotic ribosome synthesis.
Subject(s)
DEAD-box RNA Helicases/chemistry , Endoribonucleases/chemistry , Exonucleases/chemistry , Exosome Multienzyme Ribonuclease Complex/ultrastructure , RNA, Ribosomal, 18S/chemistry , Ribosomes/ultrastructure , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/genetics , Binding Sites , Cryoelectron Microscopy , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Endoribonucleases/genetics , Endoribonucleases/metabolism , Exonucleases/genetics , Exonucleases/metabolism , Exosome Multienzyme Ribonuclease Complex/genetics , Exosome Multienzyme Ribonuclease Complex/metabolism , Models, Molecular , Protein Binding , Protein Biosynthesis , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , RNA Stability , RNA, Ribosomal, 18S/genetics , RNA, Ribosomal, 18S/metabolism , Ribosomes/genetics , Ribosomes/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells1. Here we use advances in cryo-electron tomography and sub-tomogram analysis2,3 to visualize the structural dynamics of translation inside the bacterium Mycoplasma pneumoniae. To interpret the functional states in detail, we first obtain a high-resolution in-cell average map of all translating ribosomes and build an atomic model for the M. pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves 13 ribosome states that differ in their conformation and composition. These recapitulate major states that were previously resolved in vitro, and reflect intermediates during active translation. On the basis of these states, we animate translation elongation inside native cells and show how antibiotics reshape the cellular translation landscapes. During translation elongation, ribosomes often assemble in defined three-dimensional arrangements to form polysomes4. By mapping the intracellular organization of translating ribosomes, we show that their association into polysomes involves a local coordination mechanism that is mediated by the ribosomal protein L9. We propose that an extended conformation of L9 within polysomes mitigates collisions to facilitate translation fidelity. Our work thus demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells.
Subject(s)
Cryoelectron Microscopy , Mycoplasma pneumoniae , Protein Biosynthesis , Ribosomal Proteins , Ribosomes , Anti-Bacterial Agents/pharmacology , Mycoplasma pneumoniae/cytology , Mycoplasma pneumoniae/drug effects , Mycoplasma pneumoniae/metabolism , Mycoplasma pneumoniae/ultrastructure , Peptide Chain Elongation, Translational/drug effects , Polyribosomes/drug effects , Polyribosomes/metabolism , Polyribosomes/ultrastructure , Protein Biosynthesis/drug effects , Ribosomal Proteins/metabolism , Ribosomal Proteins/ultrastructure , Ribosomes/drug effects , Ribosomes/metabolism , Ribosomes/ultrastructureABSTRACT
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.
Subject(s)
Homeodomain Proteins/genetics , Protein Biosynthesis/genetics , RNA, Ribosomal/ultrastructure , Ribosomes/genetics , 5' Untranslated Regions/genetics , Gene Expression Regulation/genetics , Genes, Homeobox/genetics , Homeodomain Proteins/ultrastructure , Nucleic Acid Conformation , RNA, Messenger/genetics , RNA, Ribosomal/genetics , Ribosomes/ultrastructure , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/ultrastructure , Species SpecificityABSTRACT
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.
Subject(s)
DNA-Directed RNA Polymerases/genetics , Molecular Chaperones/genetics , Ribosomal Proteins/genetics , Ribosomes/genetics , Binding Sites/genetics , Cryoelectron Microscopy , Escherichia coli/genetics , Escherichia coli/ultrastructure , Escherichia coli Proteins/genetics , Escherichia coli Proteins/ultrastructure , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/ultrastructure , Protein Biosynthesis/genetics , RNA Folding/genetics , RNA, Ribosomal/genetics , RNA, Ribosomal/ultrastructure , Ribosomal Proteins/ultrastructure , Ribosomes/ultrastructure , Transcriptional Elongation Factors/chemistry , Transcriptional Elongation Factors/genetics , Transcriptional Elongation Factors/ultrastructureABSTRACT
Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structures with nanometre resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations in that they visualize only a single slice or a relatively small volume of the cell, respectively. Focused ion beam-scanning electron microscopy (FIB-SEM) has demonstrated the ability to image small volumes of cellular samples with 4-nm isotropic voxels1. Owing to advances in the precision and stability of FIB milling, together with enhanced signal detection and faster SEM scanning, we have increased the volume that can be imaged with 4-nm voxels by two orders of magnitude. Here we present a volume EM atlas at such resolution comprising ten three-dimensional datasets for whole cells and tissues, including cancer cells, immune cells, mouse pancreatic islets and Drosophila neural tissues. These open access data (via OpenOrganelle2) represent the foundation of a field of high-resolution whole-cell volume EM and subsequent analyses, and we invite researchers to explore this atlas and pose questions.