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In all domains of life, selenocysteine (Sec) is delivered to the ribosome by selenocysteine-specific tRNA (tRNA Sec ) with the help of a specialized translation factor, SelB in bacteria. Sec-tRNA Sec recodes a UGA stop codon next to a downstream mRNA stem–loop. Here we present the structures of six intermediates on the pathway of UGA recoding in Escherichia coli by single-particle cryo-electron microscopy. The structures explain the specificity of Sec-tRNA Sec binding by SelB and show large-scale rearrangements of Sec-tRNA Sec . Upon initial binding of SelB–Sec-tRNA Sec to the ribosome and codon reading, the 30S subunit adopts an open conformation with Sec-tRNA Sec covering the sarcin–ricin loop (SRL) on the 50S subunit. Subsequent codon recognition results in a local closure of the decoding site, which moves Sec-tRNA Sec away from the SRL and triggers a global closure of the 30S subunit shoulder domain. As a consequence, SelB docks on the SRL, activating the GTPase of SelB. These results reveal how codon recognition triggers GTPase activation in translational GTPases. The structures of several states on the pathway of SelB-mediated delivery of selenocysteine-specific tRNA to the ribosome in Escherichia coli reveal the mechanism of UGA stop codon recoding to selenocysteine and show how codon recognition triggers activation of translational GTPases. GTPase activation of elongation factor SelB In some messenger RNAs, the UGA stop codon is recoded using the specialized amino acid selenocysteine (Sec), bound to Sec-specific tRNA (tRNA Sec ). The recoding process also requires the SelB GTPase. Holger Stark and colleagues have solved multiple structures of the Escherichia coli ribosome bound to Sec-tRNA Sec and SelB to understand how SelB interacts with the tRNA, and how this complex reorders both the small and large subunits of the ribosome upon binding. The way in which these events at the codon activate the GTPase is revealed.

Crystal structures of prokaryotic ribosomes have described in detail the universally conserved core of the translation mechanism. However, many facets of the translation process in eukaryotes are not shared with prokaryotes. The crystal structure of the yeast 80S ribosome determined at 4.15 angstrom resolution reveals the higher complexity of eukaryotic ribosomes, which are 40% larger than their bacterial counterparts. Our model shows how eukaryote-specific elements considerably expand the network of interactions within the ribosome and provides insights into eukaryote-specific features of protein synthesis. Our crystals capture the ribosome in the ratcheted state, which is essential for translocation of mRNA and transfer RNA (tRNA), and in which the small ribosomal subunit has rotated with respect to the large subunit. We describe the conformational changes in both ribosomal subunits that are involved in ratcheting and their implications in coordination between the two associated subunits and in mRNA and tRNA translocation.

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. A high-resolution structure of the human ribosome determined by cryo-electron microscopy visualizes numerous RNA modifications that are concentrated at functional sites with an extended shell, and suggests the possibility of designing more specific ribosome-targeting drugs. Mapping modifications in the 80S The two subunits of the ribosome are each anchored by a large RNA molecule. After their transcription, many of the nucleotides in these ribosomal RNAs (rRNAs) are modified. The importance of these modifications is reflected in the fact that mutations in them are the basis of many diseases. Bruno Klaholz and colleagues have determined a structure of the human ribosome that has sufficient resolution to map more than 130 rRNA modifications, some of which were unknown. Comparison to sites of modification in the prokaryotic ribosome suggests how additional modifications enable stabilization of the larger eukaryotic complex. These data will expand our understanding of disease mechanisms and may suggest new therapeutic strategies.

Ribosomes are translational machineries that catalyse protein synthesis. Ribosome structures from various species are known at the atomic level, but obtaining the structure of the human ribosome has remained a challenge; efforts to address this would be highly relevant with regard to human diseases. Here we report the near-atomic structure of the human ribosome derived from high-resolution single-particle cryo-electron microscopy and atomic model building. The structure has an average resolution of 3.6 Å, reaching 2.9 Å resolution in the most stable regions. It provides unprecedented insights into ribosomal RNA entities and amino acid side chains, notably of the transfer RNA binding sites and specific molecular interactions with the exit site tRNA. It reveals atomic details of the subunit interface, which is seen to remodel strongly upon rotational movements of the ribosomal subunits. Furthermore, the structure paves the way for analysing antibiotic side effects and diseases associated with deregulated protein synthesis. The structure of the human ribosome at high resolution has been solved; by combining single-particle cryo-EM and atomic model building, local resolution of 2.9 Å was achieved within the most stable areas of the structure. The human ribosome in detail This paper presents the near-atomic structure of the human ribosome determined using single-particle cryo-electron microscopy and atomic model building. The structure reaches the high resolution of 2.9 Å in the most stable regions of the complex, allowing the visualization of previously inaccessible elements, such as regions of the ribosomal RNA scaffolding and amino acid side chains. In addition, the significant remodelling of the interface between the large and small subunits is clarified.

The ribosome is a molecular machine responsible for protein synthesis and a major target for small-molecule inhibitors. Compared to the wealth of structural information available on ribosome-targeting antibiotics in bacteria, our understanding of the binding mode of ribosome inhibitors in eukaryotes is currently limited. Here we used X-ray crystallography to determine 16 high-resolution structures of 80S ribosomes from Saccharomyces cerevisiae in complexes with 12 eukaryote-specific and 4 broad-spectrum inhibitors. All inhibitors were found associated with messenger RNA and transfer RNA binding sites. In combination with kinetic experiments, the structures suggest a model for the action of cycloheximide and lactimidomycin, which explains why lactimidomycin, the larger compound, specifically targets the first elongation cycle. The study defines common principles of targeting and resistance, provides insights into translation inhibitor mode of action and reveals the structural determinants responsible for species selectivity which could guide future drug development. Whereas previous structural investigation of ribosome inhibitors has been done using the prokaryotic ribosome, this work presents X-ray crystal structures of the yeast ribosome in complex with 16 inhibitors including eukaryotic-specific inhibitors; the inhibitors all bind the mRNA or tRNA binding sites, larger molecules appear to target specifically the first elongation cycle. Mechanisms of eukaryotic ribosome inhibition As the ribosome is a common target of antibiotics, there is a wealth of structural data on the binding of the bacterial ribosome to various inhibitors. Our understanding of inhibitor binding to the larger eukaryotic ribosome is limited. Marat Yusupov and colleagues present the structure of the yeast 80S ribosome bound to 12 eukaryote-specific and 4 broad-spectrum inhibitors. On the basis of structural data and kinetic studies, the authors propose a model for the action of cycloheximide and lactimidomycin that demonstrates that the size of an inhibitor can dictate its accessibility to the ribosome and thus its mechanism of action. This new model suggests general principles for structure-based design of new antibiotics as well as therapeutics against fungal and protozoan infections, cancers and genetic disorders induced by premature stop codons.

The initiation of bacterial translation involves the tightly regulated joining of the 50S ribosomal subunit to an initiator transfer RNA (fMet-tRNA fMet )-containing 30S ribosomal initiation complex to form a 70S initiation complex, which subsequently matures into a 70S elongation-competent complex. Rapid and accurate formation of the 70S initiation complex is promoted by initiation factors, which must dissociate from the 30S initiation complex before the resulting 70S elongation-competent complex can begin the elongation of translation 1 . Although comparisons of the structures of the 30S 2 – 5 and 70S 4 , 6 – 8 initiation complexes have revealed that the ribosome, initiation factors and fMet-tRNA fMet can acquire different conformations in these complexes, the timing of conformational changes during formation of the 70S initiation complex, the structures of any intermediates formed during these rearrangements, and the contributions that these dynamics might make to the mechanism and regulation of initiation remain unknown. Moreover, the absence of a structure of the 70S elongation-competent complex formed via an initiation-factor-catalysed reaction has precluded an understanding of the rearrangements to the ribosome, initiation factors and fMet-tRNA fMet that occur during maturation of a 70S initiation complex into a 70S elongation-competent complex. Here, using time-resolved cryogenic electron microscopy 9 , we report the near-atomic-resolution view of how a time-ordered series of conformational changes drive and regulate subunit joining, initiation factor dissociation and fMet-tRNA fMet positioning during formation of the 70S elongation-competent complex. Our results demonstrate the power of time-resolved cryogenic electron microscopy to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology. A time-resolved cryo-electron microscopy approach is used to visualize, at near-atomic resolution and on a sub-second timescale, short-lived intermediate states of a fundamental biomolecular reaction.

Ribosome-associated quality control (RQC) provides a rescue pathway for eukaryotic cells to process faulty proteins after translational stalling of cytoplasmic ribosomes 1 – 6 . After dissociation of ribosomes, the stalled tRNA-bound peptide remains associated with the 60S subunit and extended by Rqc2 by addition of C-terminal alanyl and threonyl residues (CAT tails) 7 – 9 , whereas Vms1 catalyses cleavage and release of the peptidyl-tRNA before or after addition of CAT tails 10 – 12 . In doing so, Vms1 counteracts CAT-tailing of nuclear-encoded mitochondrial proteins that otherwise drive aggregation and compromise mitochondrial and cellular homeostasis 13 . Here we present structural and functional insights into the interaction of Saccharomyces cerevisiae Vms1 with 60S subunits in pre- and post-peptidyl-tRNA cleavage states. Vms1 binds to 60S subunits with its Vms1-like release factor 1 (VLRF1), zinc finger and ankyrin domains. VLRF1 overlaps with the Rqc2 A-tRNA position and interacts with the ribosomal A-site, projecting its catalytic GSQ motif towards the CCA end of the tRNA, its Y285 residue dislodging the tRNA A73 for nucleolytic cleavage. Moreover, in the pre-state, we found the ABCF-type ATPase Arb1 in the ribosomal E-site, which stabilizes the delocalized A73 of the peptidyl-tRNA and stimulates Vms1-dependent tRNA cleavage. Our structural analysis provides mechanistic insights into the interplay of the RQC factors Vms1, Rqc2 and Arb1 and their role in the protection of mitochondria from the aggregation of toxic proteins. Cryo-electron microscopy structures of the yeast 60S ribosomal subunit in complex with Vms1 provides insights into the roles of proteins in the ribosome-associated quality control pathway.

Protein biosynthesis on the ribosome requires repeated cycles of ratcheting, which couples rotation of the two ribosomal subunits with respect to each other, and swiveling of the head domain of the small subunit. However, the molecular basis for how the two ribosomal subunits rearrange contacts with each other during ratcheting while remaining stably associated is not known. Here, we describe x-ray crystal structures of the intact Escherichia coli ribosome, either in the apo-form (3.5 angstrom resolution) or with one (4.0 angstrom resolution) or two (4.0 angstrom resolution) anticodon stem-loop tRNA mimics bound, that reveal intermediate states of intersubunit rotation. In the structures, the interface between the small and large ribosomal subunits rearranges in discrete steps along the ratcheting pathway. Positioning of the head domain of the small subunit is controlled by interactions with the large subunit and with the tRNA bound in the peptidyl-tRNA site. The intermediates observed here provide insight into how tRNAs move into the hybrid state of binding that precedes the final steps of mRNA and tRNA translocation.

Mitochondrial ribosomes (mitoribosomes) synthesize proteins encoded within the mitochondrial genome that are assembled into oxidative phosphorylation complexes. Thus, mitoribosome biogenesis is essential for ATP production and cellular metabolism 1 . Here we used cryo-electron microscopy to determine nine structures of native yeast and human mitoribosomal small subunit assembly intermediates, illuminating the mechanistic basis for how GTPases are used to control early steps of decoding centre formation, how initial rRNA folding and processing events are mediated, and how mitoribosomal proteins have active roles during assembly. Furthermore, this series of intermediates from two species with divergent mitoribosomal architecture uncovers both conserved principles and species-specific adaptations that govern the maturation of mitoribosomal small subunits in eukaryotes. By revealing the dynamic interplay between assembly factors, mitoribosomal proteins and rRNA that are required to generate functional subunits, our structural analysis provides a vignette for how molecular complexity and diversity can evolve in large ribonucleoprotein assemblies. The structures of both human and yeast mitochondrial ribosomal small subunits undergoing assembly are uncovered.

Protein synthesis in all cells is carried out by macromolecular machines called ribosomes. Although the structures of prokaryotic, yeast and protist ribosomes have been determined, the more complex molecular architecture of metazoan 80S ribosomes has so far remained elusive. Here we present structures of Drosophila melanogaster and Homo sapiens 80S ribosomes in complex with the translation factor eEF2, E-site transfer RNA and Stm1-like proteins, based on high-resolution cryo-electron-microscopy density maps. These structures not only illustrate the co-evolution of metazoan-specific ribosomal RNA with ribosomal proteins but also reveal the presence of two additional structural layers in metazoan ribosomes, a well-ordered inner layer covered by a flexible RNA outer layer. The human and Drosophila ribosome structures will provide the basis for more detailed structural, biochemical and genetic experiments. High-resolution cryo-EM density maps are used to present the structures of Drosophila and human 80S ribosomes in complex with eEF2, E-site transfer RNA and Stm1-like proteins, and reveal the presence of two additional structural layers in the ribosomes of metazoan eukaryotes. Human and Drosophila 80S ribosome structures The structures of several bacterial and yeast ribosomes have been published in the past decade, but we have had to wait for those of the much larger and more complicated metazoan ribosomes. Now Roland Beckmann and colleagues present the cryo-electron-microscopy structures of both Drosophila and human 80S ribosomes. The increased complexity appears to result in additional layers of structure. These structures will drive experiments to understand the functional and evolutionary importance of these additions.