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Translation initiation defines the identity and quantity of a synthesized protein. The process is dysregulated in many human diseases1,2. A key commitment step is when the ribosomal subunits join at a translation start site on a messenger RNA to form a functional ribosome. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when theuniversally conserved eukaryotic initiation factors eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we visualized initiation complexes that contained both eIF1A and eIF5B using single-particle cryo-electron microscopy. The resulting structure revealed how eukaryote-specific contacts between the two proteins remodel the initiation complex to orient the initiator aminoacyl-tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during translation initiation in humans.

Protein-protein interactions play critical roles in biology, but the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions not yet identified. We take advantage of advances in proteome-wide amino acid coevolution analysis and deep-learning-based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes within the Saccharomyces cerevisiae proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.3 million pairs of yeast proteins, identify 1,505 likely to interact, and build structure models for 106 previously unidentified assemblies and 806 that have not been structurally characterized. These complexes, which have as many as 5 subunits, play roles in almost all key processes in eukaryotic cells and provide broad insights into biological function.

Joining of the ribosomal subunits at a translation start site on a messenger RNA during initiation commits the ribosome to synthesize a protein. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when universally-conserved eukaryotic initiation factors (eIFs) eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we examined initiation complexes that contained both eIF1A and eIF5B using single-particle electron cryo-microscopy. The resulting structure illuminated how eukaryote-specific contacts between eIF1A and eIF5B remodel the initiation complex to orient initiator tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during human translation initiation.

Bacterial transposons are pervasive mobile genetic elements that use distinct DNA-binding proteins for horizontal transmission. For example, Escherichia coli Tn7 homes to a specific attachment site using TnsD

Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements1,2,3. Type I CRISPR–Cas systems typically target foreign DNA for degradation via joint action of the ribonucleoprotein complex Cascade and the helicase–nuclease Cas34,5, but nuclease-deficient type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons6,7. How CRISPR- and transposon-associated machineries collaborate during DNA targeting and insertion remains unknown. Here we describe structures of a TniQ–Cascade complex encoded by the Vibrio cholerae Tn6677 transposon using cryo-electron microscopy, revealing the mechanistic basis of this functional coupling. The cryo-electron microscopy maps enabled de novo modelling and refinement of the transposition protein TniQ, which binds to the Cascade complex as a dimer in a head-to-tail configuration, at the interface formed by Cas6 and Cas7 near the 3′ end of the CRISPR RNA (crRNA). The natural Cas8–Cas5 fusion protein binds the 5′ crRNA handle and contacts the TniQ dimer via a flexible insertion domain. A target DNA-bound structure reveals critical interactions necessary for protospacer-adjacent motif recognition and R-loop formation. This work lays the foundation for a structural understanding of how DNA targeting by TniQ–Cascade leads to downstream recruitment of additional transposase proteins, and will guide protein engineering efforts to leverage this system for programmable DNA insertions in genome-engineering applications., Part of this work was performed at the Simons Electron Microscopy Center and National Resource for Automated Molecular Microscopy located at the New York Structural Biology Center, supported by grants from the Simons Foundation (SF349247), NYSTAR and the NIH National Institute of General Medical Sciences (GM103310).

Significance SARS-CoV-2 is the causative agent of the COVID-19 pandemic. A molecular framework for how the virus manipulates host cellular machinery to facilitate infection is needed. Here, we integrate biochemical and single-molecule strategies to reveal molecular insight into how NSP1 from SARS-CoV-2 inhibits translation initiation. NSP1 directly binds to the small (40S) subunit of the human ribosome, which is modulated by human initiation factors. Further, NSP1 and mRNA compete with each other to bind the ribosome. Our findings suggest that the presence of NSP1 on the small ribosomal subunit prevents proper accommodation of the mRNA. How this competition disrupts the many steps of translation initiation is an important target for future studies., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a beta-CoV that recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic. A molecular framework of how the virus manipulates host cellular machinery to facilitate infection remains unclear. Here, we focus on SARS-CoV-2 NSP1, which is proposed to be a virulence factor that inhibits protein synthesis by directly binding the human ribosome. We demonstrate biochemically that NSP1 inhibits translation of model human and SARS-CoV-2 messenger RNAs (mRNAs). NSP1 specifically binds to the small (40S) ribosomal subunit, which is required for translation inhibition. Using single-molecule fluorescence assays to monitor NSP1–40S subunit binding in real time, we determine that eukaryotic translation initiation factors (eIFs) allosterically modulate the interaction of NSP1 with ribosomal preinitiation complexes in the absence of mRNA. We further elucidate that NSP1 competes with RNA segments downstream of the start codon to bind the 40S subunit and that the protein is unable to associate rapidly with 80S ribosomes assembled on an mRNA. Collectively, our findings support a model where NSP1 proteins from viruses in at least two subgenera of beta-CoVs associate with the open head conformation of the 40S subunit to inhibit an early step of translation, by preventing accommodation of mRNA within the entry channel.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

SARS-CoV-2 recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic. A molecular framework of how the virus manipulates host cellular machinery to facilitate infection remains unclear. Here, we focus on SARS-CoV-2 NSP1, which is proposed to be a virulence factor that inhibits protein synthesis by directly binding the human ribosome. Using extract-based and reconstitution experiments, we demonstrate that NSP1 inhibits translation initiation on model human and SARS-CoV-2 mRNAs. NSP1 also specifically binds to the small (40S) ribosomal subunit, which is required for translation inhibition. Using single-molecule fluorescence assays to monitor NSP1–40S subunit binding in real time, we demonstrate that eukaryotic translation initiation factors (eIFs) modulate the interaction: NSP1 rapidly and stably associates with most ribosomal pre-initiation complexes in the absence of mRNA, with particular enhancement and inhibition by eIF1 and eIF3j, respectively. Using model mRNAs and an inter-ribosomal-subunit FRET signal, we elucidate that NSP1 competes with RNA segments downstream of the start codon to bind the 40S subunit and that the protein is unable to associate rapidly with 80S ribosomes assembled on an mRNA. Collectively, our findings support a model where NSP1 associates with the open head conformation of the 40S subunit to inhibit an early step of translation, by preventing accommodation of mRNA within the entry channel.SIGNIFICANCE STATEMENTSARS-CoV-2 is the causative agent of the COVID-19 pandemic. A molecular framework for how SARS-CoV-2 manipulates host cellular machinery to facilitate infection is needed. Here, we integrate biochemical and single-molecule strategies to reveal molecular insight into how NSP1 from SARS-CoV-2 inhibits translation initiation. NSP1 directly binds to the small (40S) subunit of the human ribosome, which is modulated by human initiation factors. Further, NSP1 and mRNA compete with each other to bind the ribosome. Our findings suggest that the presence of NSP1 on the small ribosomal subunit prevents proper accommodation of the mRNA. How this competition disrupts the many steps of translation initiation is an important target for future studies.

RNA viruses are pervasive entities in the biosphere with significant impact in human health and economically important livestock. As strict cellular parasites, RNA viruses abuse host resources, redirecting them towards viral replication needs. Taking control of the cellular apparatus for protein production is a requirement for virus progression and diverse strategies of cellular mimicry and/or ribosome hijacking evolved to ensure this control. Especially in complex eukaryotes, translation is a sophisticated process, with multiple mechanisms acting on ribosomes and mRNAs. The initiation stage of translation is specially regulated, involving multiple steps and the engagement of numerous initiation factors some of them of high complexity. The use of structured RNA sequences, called Internal Ribosomal Entry Sites (IRES), in viral RNAs is a widespread strategy for the exploitation of eukaryotic initiation. Using a combination of electron cryo-microscopy (cryo-EM) and reconstituted translation initiation assays with native components, we characterized how a novel IRES at the 5’-UTR of a viral RNA assembles a functional translation initiation complex via an uAUG intermediate, redirecting the cellular machinery for protein production towards viral messengers. The IRES features a novel extended, multi-domain architecture, circling the 40S head, leveraging ribosomal sites not previously described to be exploited by any IRES. The structures and accompanying functional data, illustrate the importance of 5’-UTR regions in translation regulation and underline the relevance of the untapped diversity of viral IRESs. Given the large number of new viruses metagenomic studies have uncovered, the quantity and diversity of mechanisms for translation hijacking encrypted in viral sequences may be seriously underestimated. Exploring this diversity could reveal novel avenues in the fight against these molecular pathogens.

© The Author(s) 2020., 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., This work was supported by the US National Institutes of Health (NIH) grants GM011378 and AI047365 to J.D.P.; a Knut and Alice Wallenberg Foundation postdoctoral scholarship to J.W. (KAW 2015.0406); the Intramural Research Program of the NIH (T.E.D.).

© 2020 the Author(s)., Translation initiation controls protein synthesis by regulating the delivery of the first aminoacyl-tRNA to messenger RNAs (mRNAs). In eukaryotes, initiation is sophisticated, requiring dozens of protein factors and 2 GTP-regulated steps. The GTPase eIF5B gates progression to elongation during the second GTP-regulated step. Using electron cryomicroscopy (cryo-EM), we imaged an in vitro initiation reaction which is set up with purified yeast components and designed to stall with eIF5B and a nonhydrolyzable GTP analog. A high-resolution reconstruction of a “dead-end” intermediate at 3.6 Å allowed us to visualize eIF5B in its ribosome-bound conformation. We identified a stretch of residues in eIF5B, located close to the γ-phosphate of GTP and centered around the universally conserved tyrosine 837 (Saccharomyces cerevisiae numbering), that contacts the catalytic histidine of eIF5B (H480). Site-directed mutagenesis confirmed the essential role that these residues play in regulating ribosome binding, GTP hydrolysis, and translation initiation both in vitro and in vivo. Our results illustrate how eIF5B transmits the presence of a properly delivered initiator aminoacyl-tRNA at the P site to the distant GTPase center through interdomain communications and underscore the importance of the multidomain architecture in translation factors to sense and communicate ribosomal states., We thank the Precision Biomolecular Characterization Facility at Columbia University for technical support and access to the CD spectrometer, which is supported by NIH Award 1S10OD025102-01.

[Note: this preprint has been peer reviewed by eLife. The decision letter after peer review, based on three reviews, follows. The decision was sent on 19 May 2019.] Summary This paper describes five cryo-EM structures of ribosomal complexes apparently representing different stages of RF2-catalyzed translation termination. The novel observations here are that the tip of domain 3 of RF2 undergoes a rearrangement from an a-helical conformation to a b-hairpin conformation during termination that likely facilitates exit of the newly synthesized protein from the ribosomal polypeptide exit tunnel and that the ribosome can undergo two thermally activated, spontaneous conformational changes, a relative rotation of the ribosomal subunits and a swiveling of the 'head' domain of the small subunit, during termination that likely facilitate dissociation of RF2 from the ribosome. These are interesting observations that significantly extend our understanding of how class I RFs and ribosome conformational changes drive important steps during termination and, as such, all three reviewers recommended publication provided the following comments are addressed adequately. Essential Revisions 1) The maps provided through the eLife system seemed to be unsharpened, as they showed very little detail. However, even after sharpening them with a B-factor of -100A2, they still did not show the expected features for their respective resolutions. My suspicion is that FREALIGN has been used to overfit the data. This should be addressed in the revision. It should be indicated whether gold-standard separation of halves of the data sets were used in the final refinements, or whether those were limited to a specific spatial frequency (like was done in the classifications). If the latter, those frequencies should also be stated in the manuscript, and they should be significantly lower than the claimed resolutions. In addition: a lot of basic cryo-EM information is missing: the authors should include: a) at least one micrograph image b) some representative 2D class averages c) local resolution maps of the five structures. Also, because the density of important parts of the maps seems to be a lot worse than the resolution claimed, it would be good to explicitly mention the local resolution of the important features discussed in the main text. d) for each structure, some zoomed-in figures with the density on top of the molecular model. These figures should be chosen as to validate the resolution claim. For example, in structures I, II and V, the RNA bases should be well separated (they do so at 3.6A), and in structures III and IV beta-strands should be well separated, and many (larger) side chains should be visible. In addition, some panels with density for the most important features of each structure should be shown. e) FSC curves between the refined PDB models and the cryo-EM maps are missing from the manuscript. These should be included. In addition, to evaluate potential overfitting of the models in the maps, for each structure, the authors should also include the FSC curves between a model that was refined in half-map1 versus half-map1, as well as the FSC curve between _thesame model versus half-map2. 2) There appear to be many self-citations, and there are also a few places where relevant citations are missing or are mis-cited. There are too many to list individually, but, just a few examples: Page 4: the only citation for the phrase "recent biophysical and biochemical findings suggest a highly dynamic series of termination events" is a Rodnina paper. There are many, earlier papers from Ehrenberg, Gonzalez, Puglisi, Green, Joseph, etc. that should be cited here. Page 5: The only citation for the sentence "By contrast, biochemical experiments showed..." is a Green paper. There are earlier papers from Ehrenberg characterizing the effects of the GGQ-->GAQ mutations on the ability of RF3 to accelerate the dissociation of class I RFs from termination complexes that should be cited here. Page 5: There's a sentence that refers to X-ray, cryo-EM, and smFRET studies, but only provides citations to two smFRET studies (Casy et al, 2018 and Sternberg et al, 2009); Page 5: Moazed and Noller, 1989 identified and characterized the P/E hybrid state, but they didn't report that a deacylated P-site tRNA 'samples' the P/E hybrid state 'via a spontaneous intersubunit rotation'--that was later work from Noller and Ha; etc. There are several other instances of missing citations or mis-citations. We would ask that the authors review their citations with an eye for excessive self-citations and for missing citations or mis-citations. In this context, "Ensemble-EM" is also cited as a specific method in the introduction (Abeyrathne et al., 2016; Loveland et al., 2017). However, this method is more commonly known as (3D) classification of cryo-EM images, and there are many older, more appropriate citations. 3) The sample imaged is a model sample generated by in vitro assembly with purified components of a termination complex. In order to mimic a bona fide termination complex, a short messenger RNA with a strong Shine-Dalgarno sequence followed by a start codon and immediately after by a stop codon was used (mRNA sequence: 5'-GGC AAG GAG GUA AAA AUG UGA AAAAAA-3'). Similar constructs were used to crystallize termination complexes in the past and it has been proven by smFRET experiments that, at least regarding ribosomal inter-subunit dynamics, this model sample behaves similarly to a real termination complex with a peptide linked to the P site tRNA. However, the nature of this model sample should be apparent for the non-specialist reader, highlighting its similarities with a real termination complex but also its possible limitations, especially regarding the "artificial" nature of having a stop codon so close to the Shine-Dalgarno sequence, a situation that never happens in real mRNAs. The authors should explicitly acknowledge this and discuss its implications in the main text. 4) The authors set up a couple of somewhat 'strawman' arguments in claiming that: (i) there are discrepancies in the X-ray, cryo-EM, and smFRET literature with regard to whether ribosomes can undergo intersubunit rotation while bound to class I RFs or whether the non-rotated conformation of the ribosome is stabilized by bound class I RFs and (ii) class I RFs are able to terminate translation and dissociate from the ribosome without the aid of RF3. In the case of (i), it is obviously possible for class I RF-bound ribosomes to undergo intersubunit rotation while still favoring the non-rotated conformation of the ribosome. Moreover, there are enough differences between the cited studies, both in terms of the experimental conditions as well as the technical limitations associated with the various experimental techniques, that it is easy to rationalize differences with regard to whether the class I RF-bound ribosomes would be expected to undergo intersubunit rotation and/or whether the researchers would have been able to capture/observe intersubunit rotation. In the case of (ii), decades of biochemistry from Buckingham, Ehrenberg, Green, and others had already demonstrated that class I RFs are able to terminate translation and dissociate from the ribosome without the aid of RF3, and that the role of RF3 in termination is to accelerate the spontaneous dissociation of the class I RFs. If the authors want to highlight discrepancies in the literature, they should frame them in the context of differences between the studies, experimental design, limitations of the approaches/techniques in the cited papers that might account for such discrepancies. Re-writing this paragraph also in the light of addressing the missing citations and mis-citations pointed out under (2) will further help in toning these arguments down, which would strengthen the manuscript's scholarship. 5) Class I RFs are post-translationally methylated at the Q residue of the GGQ motif of domain 3 and Buckingham, Ehrenberg, and others have shown that this methylation accelerates and/or facilitates class I-catalyzed termination both in vitro and in vivo. Nonetheless, Svidritskiy et al do not report whether and to what extent their RF2 is methylated. Was RF2 overexpressed in a manner that ensured homogeneous methylation or lack of methylation? If they are overexpressing prfB and not overexpressing prmC, it is likely that they have a mix of methylated and unmethylated RF2. Assuming they are using the wt E. coli prfB gene, then the residue at position 246 is a T, rather than an A or S, and Buckingham has shown that, in the wt T246 background, a lack of methylation at Q252 is either seriously detrimental in richer media or lethal in more minimal media. It was felt that a discussion of this issue was not needed in the main text, but that it would be helpful if the authors would include the important/relevant experimental details in the Methods section, for example, did they use the T246 wt E. coli variant of RF2; and did they overexpress prmC along with prfB? 6) Structure I is denoted and treated as a pre-termination complex, but that does not seem at all possible given that the sample was prepared by incubating a pre-termination complex for 30 min in the presence of excess RF2, conditions that Figure 1-Figure Supplement 3 suggest results in robust termination. Structure I is more likely the non-rotated conformation of a post-termination complex that is in equilibrium with its rotated counterpart, Structure V. Based on my reading of the manuscript, it is likely that the authors understand this point, but are nonetheless using this structure as a mimic/analog of a pre-termination complex. If so, I think this is fine, but the authors should explicitly state that this is what they are doing. Related to this, the authors should clarify the description of their activity assay, show the raw data, and/or report 'Released [S35]-fMet (%)' instead of 'Released [S35]-fMet, CPM' on the y-axis of Figure 1-Figure Supplement 3; as the activity assay is currently described, reported, and plotted, it is impossible to determine whether RF2 is 1% or 99% active in termination. 7) The final sentence of the manuscript reads: "Translation termination and recycling of the release factors and the ribosome therefore rely on the spontaneous ribosome dynamics, triggered by local rearrangements of the universally conserved elements of the peptidyl-transferase and decoding centers". There are a couple of problems with this sentence as written. First, smFRET experiments by Gonzalez, Puglisi, and Rodnina have previously shown that "Translation termination and recycling of the release factors and the ribosome therefore rely on the spontaneous ribosome dynamics" and the relevant articles should therefore be cited here. Moreover, given the data are static structures solved using a sample that is at equilibrium, it is not clear how the authors determined that these spontaneous ribosome dynamics were "triggered by local rearrangements of the universally conserved elements of the peptidyl-transferase and decoding centers". Isn't it equally possible, given the data presented, that the local rearrangements were triggered by the ribosome dynamics?

TheColonyCollapseDisorder or CCD is a multi-faceted syndrome decimating bee populations worldwide[1]. A group of viruses of the widely distributedDicistroviridaefamily have been identified as a causing agent of CCD[2]. This family of viruses employ non-coding RNA sequences, calledInternalRibosomalEntrySite (IRES), to precisely exploit the host machinery for protein production. Using single-particle cryo-electron microscopy (cryo-EM) we have characterized at high resolution how the IRES of the intergenic region of theIsraeliAcuteParalysisVirus (IAPV) captures and redirects translating ribosomes towards viral messengers. Through a series of six structures at nominal resolutions close to 3Å, we could reconstruct the trajectory of IAPV-IRES from an early small subunit recruitment to a final post-translocated state in the ribosome. An early commitment of IRES/ribosome complexes for global pre-translocation mimicry explains the high efficiency observed for this IRES. The presented structures will help guide on-going efforts directed towards fighting CCD through RNA-interference technology [3].

In order to survive, bacteria continually sense, and respond to, environmental fluctuations. Stringent control represents a key bacterial stress response to nutrient starvation that leads to rapid and comprehensive reprogramming of metabolic and transcriptional patterns. In general, transcription of genes for growth and proliferation is downregulated, while those important for survival and virulence are upregulated. Amino acid starvation is sensed by depletion of the aminoacylated tRNA pools, and this results in accumulation of ribosomes stalled with non-aminoacylated (uncharged) tRNA in the ribosomal A site. RelA is recruited to stalled ribosomes and activated to synthesize a hyperphosphorylated guanosine analogue, (p)ppGpp, which acts as a pleiotropic secondary messenger. However, structural information about how RelA recognizes stalled ribosomes and discriminates against aminoacylated tRNAs is missing. Here we present the cryo-electron microscopy structure of RelA bound to the bacterial ribosome stalled with uncharged tRNA. The structure reveals that RelA utilizes a distinct binding site compared to the translational factors, with a multi-domain architecture that wraps around a highly distorted A-site tRNA. The TGS (ThrRS, GTPase and SpoT) domain of RelA binds the CCA tail to orient the free 3' hydroxyl group of the terminal adenosine towards a β-strand, such that an aminoacylated tRNA at this position would be sterically precluded. The structure supports a model in which association of RelA with the ribosome suppresses auto-inhibition to activate synthesis of (p)ppGpp and initiate the stringent response. Since stringent control is responsible for the survival of pathogenic bacteria under stress conditions, and contributes to chronic infections and antibiotic tolerance, RelA represents a good target for the development of novel antibacterial therapeutics.

Co-opting the cellular machinery for protein production is a compulsory requirement for viruses. The Cricket Paralysis Virus employs an Internal Ribosomal Entry Site (CrPV-IRES) to express its structural genes in the late stage of infection. Ribosome hijacking is achieved by a sophisticated use of molecular mimicry to tRNA and mRNA, employed to manipulate intrinsically dynamic components of the ribosome. Binding and translocation through the ribosome is required for this IRES to initiate translation. We report two structures, solved by single particle electron cryo-microscopy (cryoEM), of a double translocated CrPV-IRES with aminoacyl-tRNA in the peptidyl site (P site) of the ribosome. CrPV-IRES adopts a previously unseen conformation, mimicking the acceptor stem of a canonical E site tRNA. The structures suggest a mechanism for the positioning of the first aminoacyl-tRNA shared with the distantly related Hepatitis C Virus IRES., eLife digest Viruses cannot replicate themselves, but instead depend on components of the host cell for their own survival. Once a virus successfully enters a cell, it must use part of the cell’s machinery – specifically the ribosomes – to produce its own proteins. Ribosomes normally make the cell’s proteins by reading instructions written in molecules known as messenger RNAs (or mRNAs for short). Viruses hijack ribosomes using structured RNA segments in its mRNAs that can mimic natural components of the cell’s protein-producing machinery. These RNA sequences, known as IRESs, feature a refined balance between rigidity and flexibility. Their flexible nature has made them difficult to study in the past, though the latest advances in electron cryo-microscopy mean that IRESs can now be directly observed in complex with ribosomes. Pisareva et al. sought to image a prototypical IRES sequence from the Cricket Paralysis Virus as it is transitioned through the ribosome. The idea was to characterize the late stages of ribosome hijacking. First, all the essential components were purified, mixed in the laboratory, and then imaged via electron cryo-microscopy. Image processing and sorting algorithms were then used to visualize the process at a high level of detail. Unexpectedly, this showed that the IRES changes shape dramatically to mimic part of another RNA molecule, a tRNA, when it reaches the so-called exit site of the ribosome. Short for transfer RNAs, tRNAs are molecules that bring the building blocks of proteins (called amino acids) to the ribosome, ready to be linked together. The shape change in the IRES is coupled with the placement of the first amino acid-loaded tRNA in a site on the ribosome that commits it to producing the viral protein. These results illustrate the remarkable ability of RNA molecules, in general, and IRES sequences, in particular, to adopt distinctive and context-specific shapes. These features seem to be widely conserved among diverse virus families as a similar shape change has been see in the IRES of the distantly related Hepatits C Virus. Together these new insights could lead to new strategies to interfere with viral replication and further studies that deepen our understanding of how ribosome and RNA-based mechanisms work generally inside cells.

Placement of an initiator aminoacyl-tRNA [(f)Met-tRNAi(f)Met] base paired with the AUG initiation codon of a messenger RNA (mRNA) is the first step of translation. The eukaryotic translation factor eIF5B or its bacerial homologue IF2 facilitate the correct positioning of initiator tRNA in the P site of the ribosome. We report the electron cryomicroscopy (cryoEM) structure of a stabilized intermediate state of a yeast 80S/tRNAiMet/eIF5B complex at 3.6 Å resolution. The structure shows how a universally conserved tyrosine couples the rotational state of the small ribosomal subunit with GTP hydrolysis.

Malaria control is heavily dependent on chemotherapeutic agents for disease prevention and drug treatment. Defining the mechanism of action for licensed drugs, for which no target is characterized, is critical to the development of their second-generation derivatives to improve drug potency towards inhibition of their molecular targets. Mefloquine is a widely used antimalarial without a known mode of action. Here, we demonstrate that mefloquine is a protein synthesis inhibitor. We solved a 3.2 Å cryo-electron microscopy structure of the Plasmodium falciparum 80S ribosome with the (+)-mefloquine enantiomer bound to the ribosome GTPase-associated centre. Mutagenesis of mefloquine-binding residues generates parasites with increased resistance, confirming the parasite-killing mechanism. Furthermore, structure-guided derivatives with an altered piperidine group, predicted to improve binding, show enhanced parasiticidal effect. These data reveal one possible mode of action for mefloquine and demonstrate the vast potential of cryo-electron microscopy to guide the development of mefloquine derivatives to inhibit parasite protein synthesis.

The bacterial stringent response links nutrient starvation with the transcriptional control of genes. This process is initiated by the stringent factor RelA, which senses the presence of deacylated tRNA in the ribosome as a symptom of amino-acid starvation to synthesize the alarmone (p)ppGpp. Here we report a cryo-EM study of RelA bound to ribosomes bearing cognate, deacylated tRNA in the A-site. The data show that RelA on the ribosome stabilizes an unusual distorted form of the tRNA, with the acceptor arm making contact with RelA and far from its normal location in the peptidyl transferase centre.

Viral mRNA sequences with a type IV IRES are able to initiate translation without any host initiation factors. Initial recruitment of the small ribosomal subunit as well as two translocation steps before the first peptidyl transfer are essential for the initiation of translation by these mRNAs. Using electron cryomicroscopy (cryo-EM) we have structurally characterized at high resolution how the Cricket Paralysis Virus Internal Ribosomal Entry Site (CrPV-IRES) binds the small ribosomal subunit (40S) and the translocation intermediate stabilized by elongation factor 2 (eEF2). The CrPV-IRES restricts the otherwise flexible 40S head to a conformation compatible with binding the large ribosomal subunit (60S). Once the 60S is recruited, the binary CrPV-IRES/80S complex oscillates between canonical and rotated states (Fernández et al., 2014; Koh et al., 2014), as seen for pre-translocation complexes with tRNAs. Elongation factor eEF2 with a GTP analog stabilizes the ribosome-IRES complex in a rotated state with an extra ~3 degrees of rotation. Key residues in domain IV of eEF2 interact with pseudoknot I (PKI) of the CrPV-IRES stabilizing it in a conformation reminiscent of a hybrid tRNA state. The structure explains how diphthamide, a eukaryotic and archaeal specific post-translational modification of a histidine residue of eEF2, is involved in translocation.

SMG-9 is part of a protein kinase complex, SMG1C, which consists of the SMG-1 kinase, SMG-8 and SMG-9. SMG1C mediated phosphorylation of Upf1 triggers nonsense-mediated mRNA decay (NMD), a eukaryotic surveillance pathway that detects and targets for degradation mRNAs harboring premature translation termination codons. Here, we have characterized SMG-9, showing that it comprises an N-terminal 180 residue intrinsically disordered region (IDR) followed by a well-folded C-terminal domain. Both domains are required for SMG-1 binding and the integrity of the SMG1C complex, whereas the C-terminus is sufficient to interact with SMG-8. In addition, we have found that SMG-9 assembles in vivo into SMG-9:SMG-9 and, most likely, SMG-8:SMG-9 complexes that are not constituents of SMG1C. SMG-9 self-association is driven by interactions between the C-terminal domains and surprisingly, some SMG-9 oligomers are completely devoid of SMG-1 and SMG-8. We propose that SMG-9 has biological functions beyond SMG1C, as part of distinct SMG-9-containing complexes. Some of these complexes may function as intermediates potentially regulating SMG1C assembly, tuning the activity of SMG-1 with the NMD machinery. The structural malleability of IDRs could facilitate the transit of SMG-9 through several macromolecular complexes. © 2010 The Author(s)., Spanish Ministry of Science and Innovation (SAF2008- 00451 to O.L ., SAF2008-00479 to J.T.); ‘Red Temática de Investigación Cooperativa en Cáncer (RTICC)’ from the ‘Instituto de Salud Carlos III’ (RD06/0020/1001 to O.L. and RD06/0020/0011 to J.T.); Autonomous Region of Madrid (CAM S-BIO-0214-2006 to O.L.); Human Frontiers Science Program (RGP39/2008 to O.L.); ‘Consejería de Educación de la Comunidad de Madrid y Fondo Social Europeo’ (to E.A.P.); Japan Society for the Promotion of Science (to A.Y. and S.O.); Japan Science and Technology Corporation (to A.Y. and S.O.); Ministry of Education, Culture, Sports, Science and Technology of Japan (to S.O.); Yokohama Foundation for Advancement of Medical Science (to A.Y.). Funding for open access charge: Spanish Ministry of Science and Innovation

10 páginas, 5 figuras, 1 tabla -- PAGS nros. 1488-1497, Human kallikrein 7, a major protease of human skin, has been synthesized directly in its native conformation in Escherichia coli by forcing the secretion of the newly synthesized polypeptide into the bacterial periplasm. The procedure yields a stable kallikrein 7 with highly specific activity that is inhibited efficiently by its specific inhibitor LEKTI domain 6. The protein was crystallized, and its three-dimensional structure was solved in the absence of protease inhibitors. The structure obtained agrees with that reported recently for human tissue kallikrein 7 crystallized in the presence of protease inhibitors from a preparation obtained in a baculovirus protein expression system. A model of the interaction between the protease and its inhibitor is proposed on the basis of both the three-dimensional structure of human tissue kallikrein 7 reported here and that of the LEKTI domain 6 solved previously by NMR, This work was funded by BFU2005-05055 from the Ministerio de Educación y Ciencia (Spain). I. S. F. is a recipient of a PhD fellowship from the Ministerio de Educación y Ciencia (Spain), and L. S. was sponsored by a Marie Curie TOK-IAP (MTCK-CT-2004-014456

The N-terminal acetylation of Sir3 is essential for heterochromatin establishment and maintenance in yeast, but its mechanism of action is unknown. The crystal structure of the N-terminally acetylated BAH domain of Saccharomyces cerevisiae Sir3 bound to the nucleosome core particle reveals that the N-terminal acetylation stabilizes the interaction of Sir3 with the nucleosome. Additionally, we present a new method for the production of protein–nucleosome complexes for structural analysis.

11 páginas, 7 figuras. Dispone de material suplementario en: t http://dx.doi.org/10.1016/j.cell.2014.10.001., During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit. This conformation (POUT) is compatible with scanning mRNA for the AUG start codon. Base pairing with AUG is thought to promote isomerization to a more stable conformation (PIN) that arrests scanning and promotes dissociation of eIF1 from the 40S subunit. Here, we present a cryoEM reconstruction of a yeast preinitiation complex at 4.0 Å resolution with initiator tRNA in the PIN state, prior to eIF1 release. The structure reveals stabilization of the codon-anticodon duplex by the N-terminal tail of eIF1A, changes in the structure of eIF1 likely instrumental in its subsequent release, and changes in the conformation of eIF2. The mRNA traverses the entire mRNA cleft and makes connections to the regulatory domain of eIF2?, eIF1A, and ribosomal elements that allow recognition of context nucleotides surrounding the AUG codon., This work was funded by grants to V.R. from the UK Medical Research Council (MC_U105184332), Wellcome Trust Senior Investigator award (WT096570), the Agouron Institute and the Jeantet Foundation, from the NIH (GM62128) previously to J.R.L., and from the Human Frontiers in Science Program (RGP-0028/2009) to A.G.H., J.R.L., and V.R., and by the Intramural Research Program of the NIH (A.G.H., P.M.-M., J.R.L., and A.M.)

Malaria inflicts an enormous burden on global human health. The emergence of parasite resistance to front-line drugs has prompted a renewed focus on the repositioning of clinically approved drugs as potential anti-malarial therapies. Antibiotics that inhibit protein translation are promising candidates for repositioning. We have solved the cryo-EM structure of the cytoplasmic ribosome from the human malaria parasite, Plasmodium falciparum, in complex with emetine at 3.2 Å resolution. Emetine is an anti-protozoan drug used in the treatment of ameobiasis that also displays potent anti-malarial activity. Emetine interacts with the E-site of the ribosomal small subunit and shares a similar binding site with the antibiotic pactamycin, thereby delivering its therapeutic effect by blocking mRNA/tRNA translocation. As the first cryo-EM structure that visualizes an antibiotic bound to any ribosome at atomic resolution, this establishes cryo-EM as a powerful tool for screening and guiding the design of drugs that target parasite translation machinery. DOI: http://dx.doi.org/10.7554/eLife.03080.001, eLife digest Each year, malaria kills more than 600,000 people, mostly children younger than 5 years old. Humans who have been bitten by mosquitoes infected with malaria-causing parasites become ill as the parasites rapidly multiply in blood cells. Although there are several drugs that are currently used to treat malaria, the parasites are rapidly developing resistance to them, setting off an urgent hunt for new malaria drugs. Developing new antimalarial medications from scratch is likely to take decades—too long to combat the current public health threat posed by emerging strains of drug-resistant parasites. To speed up the process, scientists are investigating whether drugs developed for other illnesses may also act as therapies for malaria, either when used alone or in combination with existing malaria drugs. Certain antibiotics—including one called emetine—have already shown promise as antimalarial drugs. These antibiotics prevent the parasites from multiplying by interfering with the ribosome—the part of a cell that builds new proteins. However, humans become ill after taking emetine for long periods because it also blocks the production of human proteins. Tweaking emetine so that it acts only against the production of parasite proteins would make it a safer malaria treatment. To do this, scientists must first map the precise interactions between the drug and the ribosomes in parasites. Wong et al. have now used a technique called cryo-electron microscopy to examine the ribosome of the most virulent form of malaria parasite. This technique uses very cold temperatures to rapidly freeze molecules, allowing scientists to look at molecular-level details without distorting the structure of the molecule—a problem sometimes encountered in other techniques. The images of the parasitic ribosome taken by Wong, Bai, Brown et al. show that emetine binds to the end of the ribosome where the instructions for how to assemble amino acids into a protein are copied from strands of RNA. In addition, the images revealed features of the parasitic ribosome that are not found in the human form. Drug makers could exploit these features to improve emetine so that it more specifically targets the production of proteins by the parasite and is less toxic to humans. DOI: http://dx.doi.org/10.7554/eLife.03080.002

Summary 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., Graphical Abstract, Highlights • The high-resolution structure of CrPV-IRES bound to the ribosome was solved by cryoEM • Pseudoknot I of CrPV-IRES binds in the decoding center, thus blocking the A site • CrPV-IRES mimics a pretranslocation rather than initiation complex of the ribosome • Translocation of CrPV-IRES by eEF2 is required for the start of translation, A high-resolution structure of the cricket paralysis virus IRES bound to the eukaryotic ribosome reveals a surprising mechanism of translation initiation that requires a translocation step.

Introduction Initiation of protein synthesis is a key step in the control of gene expression. In eukaryotes, initiation is a highly complex process that requires almost a dozen protein factors. The last step involves joining of the large and small subunits of the ribosome to form the 80 S initiation complex with the transfer RNA (tRNA) in the P-site base paired to the start codon. This step is catalyzed by the guanosine triphosphatase (GTPase) factor eIF5B. In addition, eIF5B is thought to play a role in ensuring that translation initiation takes place only on mature ribosomes. Methods The development of fast, direct electron detectors and new methods of image analysis for cryo–electron microscopy allow high-resolution reconstructions from much smaller numbers of particles than previously possible. We used these new methods to provide feedback to improve the biochemical preparation of samples for structure determination of the eukaryotic translation initiation complex with initiator tRNA and eIF5B, which was trapped on the ribosome with the nonhydrolyzable GTP analog GDPCP. Results Although the structure of the fully assembled complex was calculated from only 5143 particles, representing just 3% of the population in the sample, it was possible to obtain a resolution of 6.6 Å. This allowed us to propose a molecular model for the initiation complex. The structure shows that the subunits of the ribosome are rotated relative to the canonical state after initiation. The long helix and C-terminal domain of eIF5B have changed conformation and moved into the ribosome, where the C-terminal domain interacts with the initiator tRNA. The tRNA is stabilized in a distorted conformation, with its 3′-CCA end out of the peptidyl transferase center. Discussion The conformational change in eIF5B may be induced upon binding to the small (40 S ) subunit when it specifically recognizes initiator tRNA. In its altered conformation, eIF5B interacts simultaneously with the initiator tRNA and the GTPase center of the ribosome, thus coupling GTP hydrolysis with tRNA recognition in the ribosome. The large number of contacts made by eIF5B with the ribosomal subunits and tRNA is consistent with its role in subunit joining. A close contact with the eukaryote-specific ribosomal protein L40 would not be possible in the immature ubiquitinated form of L40, thus precluding the recruitment of immature 60 S subunits. A comparison with previous work on the bacterial homolog IF2 suggests that the mechanism of this step of initiation is conserved across kingdoms. Finally, the use of recent advances in cryo-EM to determine a relatively high-resolution structure of the eIF5B-ribosome complex from a very small fraction of a sample could be a general approach for the study of other dynamic or transient biological complexes.

Introduction After peptidyl transfer, the movement of messenger RNA (mRNA) and transfer RNAs (tRNAs) with respect to the ribosome places the next mRNA codon in the A site. This process of translocation proceeds via an intermediate state in which the acceptor ends of the tRNAs have moved with respect to the 50S subunit but not the 30S subunit, to result in A/P and P/E tRNA hybrid states. The guanosine triphosphatase elongation factor G (EF-G) catalyzes the subsequent movement of mRNA and tRNA with respect to the 30S subunit. How EF-G binds to the intermediate state of the ribosome and how this results in guanosine 5′-triphosphate (GTP) hydrolysis and translocation are questions that will be greatly facilitated by a high-resolution structure of the complex. Structure of EF-G with GDPCP bound to the ribosome in an intermediate state of translocation. (A) Overview of the structure with a hybrid P/E tRNA. (B) Rotation of the body and swiveling of the head of the 30S subunit (yellow), compared to the canonical state (gray). (C) Catalytic site showing conserved residues around the GDPCP molecule stabilized in an activated conformation. Methods Thermus thermophilus ribosomes lacking protein L9 were crystallized in an intermediate state with mRNA, a hybrid P/E tRNA, and EF-G with the nonhydrolyzable GTP analog GDPCP. The structure was solved by molecular replacement and refined to 2.9 A resolution. Results The 50S and 30S ribosomal subunits are rotated relative to each other, as was expected from previous cryo–electron microscopy studies. The L1 stalk on the 50S subunit moves inward to stabilize the P/E hybrid-state tRNA, and atomic details of this interaction can now be seen. Domain IV of EF-G takes up an orientation intermediate between that of the isolated form of EF-G and that of EF-G bound to the ribosome in the fully translocated state. The catalytic center of EF-G shows that key switch regions surrounding the γ-phosphate of GDPCP are clearly visible and therefore ordered. Moreover, key conserved residues in EF-G, including a histidine and an aspartate, change conformation relative to both the isolated and fully translocated structure. These activated conformations appear to be stabilized by the highly conserved sarcin-ricin RNA loop (SRL) of the 50S subunit. Discussion Comparison with the posttranslocational state suggests that interactions between the tRNA and L1 stalk are preserved throughout translocation and that these are probably an essential feature of translocation required for stabilization of the hybrid P/E state. In the isolated structure of EF-G, domain IV, because of its orientation, would largely avoid a clash with A-site tRNA, as would be required for formation of a transient initial complex. In our structure, domain IV partly extends into the A site, which is consistent with the observation that EF-G facilitates translocation at a slow rate even without GTP hydrolysis. The catalytic center of EF-G has essentially the same structure as that previously observed for elongation factor Tu (EF-Tu), with the highly conserved histidine stabilized by the SRL in an orientation that coordinates a water molecule in position for hydrolysis of GTP. This shows that although EF-Tu and EF-G bind to very different states of the ribosome, the mechanism of activation of GTP hydrolysis is probably the same for these two factors, and possibly for other translational GTPases.

In vitro mitogenesis assays have shown that sulfated glycosaminoglycans (GAGs; heparin and heparan sulfate) cause an enhancement of the mitogenic activity of fibroblast growth factors (FGFs). Herein, we report that the simultaneous presence of FGF and the GAG is not an essential requisite for this event to take place. Indeed, preincubation with heparin (just before FGF addition) of cells lacking heparan sulfate produced an enhancing effect equivalent to that observed when the GAG and the protein are simultaneously added. A first structural characterization of this effect by analytical ultracentrifugation of a soluble preparation of the heparin-binding domain of fibroblast growth factor receptor 2 (FGFR2) and a low molecular weight (3 kDa) heparin showed that the GAG induces dimerization of FGFR2. To derive a high resolution structural picture of this molecular recognition process, the interactions of a soluble heparin-binding domain of FGFR2 with two different homogeneous, synthetic, and mitogenically active sulfated GAGs were analyzed by NMR spectroscopy. These studies, assisted by docking protocols and molecular dynamics simulations, have demonstrated that the interactions of these GAGs with the soluble heparin-binding domain of FGFR induces formation of an FGFR dimer; its architecture is equivalent to that in one of the two distinct crystallographic structures of FGFR in complex with both heparin and FGF1. This preformation of the FGFR dimer (with similar topology to that of the signaling complex) should favor incorporation of the FGF component to form the final assemblage of the signaling complex, without major entropy penalty. This cascade of events is probably at the heart of the observed activating effect of heparin in FGF-driven mitogenesis.

Although electron cryo-microscopy (cryo-EM) single-particle analysis has become an important tool for structural biology of large and flexible macro-molecular assemblies, the technique has not yet reached its full potential. Besides fundamental limits imposed by radiation damage, poor detectors and beam-induced sample movement have been shown to degrade attainable resolutions. A new generation of direct electron detectors may ameliorate both effects. Apart from exhibiting improved signal-to-noise performance, these cameras are also fast enough to follow particle movements during electron irradiation. Here, we assess the potentials of this technology for cryo-EM structure determination. Using a newly developed statistical movie processing approach to compensate for beam-induced movement, we show that ribosome reconstructions with unprecedented resolutions may be calculated from almost two orders of magnitude fewer particles than used previously. Therefore, this methodology may expand the scope of high-resolution cryo-EM to a broad range of biological specimens. DOI: http://dx.doi.org/10.7554/eLife.00461.001, eLife digest Determining the structure of proteins and other biomolecules at the atomic level is central to understanding many aspects of biology. X-ray crystallography is the best-known technique for structural biology but, as the name suggests, it works only with samples that can be crystallized. Electron cryo-microscopy (cryo-EM) could, potentially, be used to determine the atomic structures of biomolecules that cannot be crystallized, but at present the resolution that can be achieved with this approach is sufficient only for imaging certain types of viruses. In cryo-EM, a solution of the biomolecule of interest is frozen in a thin layer of ice, and this layer is imaged in an electron microscope. By combining images of many identical biomolecules in many different orientations, it is possible to work backwards and determine their 3D structure. However, in order to determine this structure at high resolution, it is necessary to make repeated measurements to reduce high levels of noise in the images. Cryo-EM images are usually recorded on a photographic film or a CCD (charge-coupled device) camera. However, photographic film is unsuitable for high-throughput methods because it has to be handled manually, while the efficiency of CCD cameras is limited because the electrons have to be converted into visible light to be detected. Digital cameras that can detect electrons directly have become available recently, and these are more efficient than both film and CCD cameras. They are also much faster, which means that it is possible to record videos of the sample during the time (typically ∼1 s) it is being exposed to the electron beam. Processing these videos could then—in theory—compensate for any movements of the biomolecules that are induced by the electron beam. Along with radiation damage caused by the electrons, these beam-induced movements have been a major limitation on the resolution that can be achieved with cryo-EM. Bai et al. demonstrate the potential of direct-electron detectors in cryo-EM by determining the structures of two ribosomes. Using a novel statistical algorithm to accurately follow the movements of the ribosomes during the time they are exposed to the electron beam, they are able to compensate for these movements, and this makes it possible to determine the structures of the ribosomes with near-atomic precision. Moreover, the resolution they achieve with just ∼30,000 ribosomes is better than that previously achieved with more than a million ribosomes, allowing small details inside the ribosome – such as ß-strands and bulky amino-acid side chains – to be resolved with cryo-EM for the first time. The work of Bai et al. could, therefore, allow researchers to use cryo-EM to determine the structure of many more biomolecules with atomic precision. DOI: http://dx.doi.org/10.7554/eLife.00461.002

37 p-.8 fig.-3 tab., The comparatively small number of members of the family of adhesion/growth-regulatory galectins in chicken predestines this system as an attractive model to study divergence of these lectins after gene duplications. Expression profiling of the three homodimeric (proto-type) chicken galectins (CG-1A, CG-1B and CG-2) has raised evidence for distinct functionalities, explaining the interest in a detailed crystallographic analysis of CG-2. As revealed here, marked differences are found in the ligand-binding site and in the contact pattern within the homodimer interface, underlying a characteristic orientation of the two subunits. Notably, a distinctive trimer of dimers that is unique in all galectin crystal structures reported to date forms the core unit of the crystallographic assembly. Combination with spectroscopic and thermodynamic measurements, and comparisons with CG-1A and CG-1B, identifies differential changes in circular-dichroism spectra in the presence of lactose, reflecting the far-reaching impact of the ligand on hydrodynamic behavior, and inter-galectin differences in both the entropy and the enthalpy of binding. This structural information is a salient step to complete the analysis of the full set of galectins from this model organism., This work has been kindly supported by grants BFU2009-10052, BFU2011-24615, BFU2012-36825 and CSD2009-00088 from the Spanish Ministry of Science and Innovation, the CIBER of Respiratory Diseases (CIBERES), an initiative from ISCIII, the Regional Government of Madrid (S2010/BMD-2353), and funding from the European Union’s Seventh Framework Program FP7/2007-2013 under REA grant agreements no. 260600 (“GlycoHIT”) and 317297 (“GLYCOPHARM”).

Here, the structure of the 30S ribosomal subunit and the 70S ribosome in complex with a messenger RNA with pseudouridine in the place of uridine reveals unexpected base pairing. When messenger RNA is translated into protein, the end of the protein-coding sequence is indicated by a three-base stop codon. Stop codons do not code for an amino acid, but recently it was shown that changing the first base to a pseudouridine (Ψ, the C-glycoside isomer of the nucleoside uridine) allows the incorporation of an amino acid, such that translation could continue past the stop codon. Venki Ramakrishnan and colleagues have determined the structure of the 30S ribosomal subunit in complex with an mRNA with ΨAG at the A site and a portion of serine transfer RNA. The structure reveals unexpected purine–purine base pairing at the first position of the codon and unusual pairing at the second and third positions. This study provides additional evidence for plasticity in the decoding centre of the ribosome. During normal translation, the binding of a release factor to one of the three stop codons (UGA, UAA or UAG) results in the termination of protein synthesis. However, modification of the initial uridine to a pseudouridine (Ψ) allows efficient recognition and read-through of these stop codons by a transfer RNA (tRNA), although it requires the formation of two normally forbidden purine–purine base pairs1. Here we determined the crystal structure at 3.1 A resolution of the 30S ribosomal subunit in complex with the anticodon stem loop of tRNASer bound to the ΨAG stop codon in the A site. The ΨA base pair at the first position is accompanied by the formation of purine–purine base pairs at the second and third positions of the codon, which show an unusual Watson–Crick/Hoogsteen geometry. The structure shows a previously unsuspected ability of the ribosomal decoding centre to accommodate non-canonical base pairs.

Summary Chlorinated compounds are important environmental pollutants whose biodegradation may be limited by inefficient dechlorinating enzymes. Dictyostelium amoebae produce a chlorinated alkyl phenone called DIF which induces stalk cell differentiation during their multicellular development. Here we describe the identification of DIF dechlorinase. DIF dechlorinase is active when expressed in bacteria, and activity is lost from Dictyostelium cells when its gene, drcA, is knocked out. It has a Km for DIF of 88 nM and Kcat of 6.7 s−1. DrcA is related to glutathione S-transferases, but with a key asparagine-to-cysteine substitution in the catalytic pocket. When this change is reversed, the enzyme reverts to a glutathione S-transferase, thus suggesting a catalytic mechanism. DrcA offers new possibilities for the rational design of bioremediation strategies., Highlights ► DIF-1 reductive dechlorinase is a GST-related protein ► A cysteine in the catalytic pocket is key for completing the reaction ► DIF dechlorination occurs through a glutathione-conjugated intermediate

Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that regulates the degradation of mRNAs harboring premature translation termination codons. NMD also influences the expression of many physiological transcripts. SMG-1 is a large kinase essential to NMD that phosphorylates Upf1, which seems to be the definitive signal triggering mRNA decay. However, the regulation of the kinase activity of SMG-1 remains poorly understood. Here, we reveal the three-dimensional architecture of SMG-1 in complex with SMG-8 and SMG-9, and the structural mechanisms regulating SMG-1 kinase. A bent arm comprising a long region of HEAT (huntington, elongation factor 3, a subunit of PP2A and TOR1) repeats at the N terminus of SMG-1 functions as a scaffold for SMG-8 and SMG-9, and projects from the C-terminal core containing the phosphatidylinositol 3-kinase domain. SMG-9 seems to control the activity of SMG-1 indirectly through the recruitment of SMG-8 to the N-terminal HEAT repeat region of SMG-1. Notably, SMG-8 binding to the SMG-1:SMG-9 complex specifically down-regulates the kinase activity of SMG-1 on Upf1 without contacting the catalytic domain. Assembly of the SMG-1:SMG-8:SMG-9 complex induces a significant motion of the HEAT repeats that is signaled to the kinase domain. Thus, large-scale conformational changes induced by SMG-8 after SMG-9-mediated recruitment tune SMG-1 kinase activity to modulate NMD. © 2011 by Cold Spring Harbor Laboratory Press., This work was funded by the Spanish Ministry of Science and Innovation (SAF2008-00451 to O.L.); the Red Temática de Investigación Cooperativa en Cáncer (RTICC) from the Instituto de Salud Carlos III (RD06/0020/1001 to O.L.); the Autonomous Region of Madrid (CAM S-BIO-0214-2006 to O.L.); the Human Frontiers Science Program (RGP39/2008 to O.L.); the Consejería de Educación de la Comunidad de Madrid y Fondo Social Europeo (to E.A.P.); the Japan Society for the Promotion of Science (to A.Y. and S.O.); the Japan Science and Technology Corporation (to A.Y. and S.O.); the Ministry of Education,Culture, Sports, Science, and Technology of Japan (to S.O.); the Takeda Science Foundation (to S.O.); and the Yokohama Foundation for Advancement of Medical Science (to A.Y.).

4 páginas, 6 figuras, 2 tablas -- PAGS nros. 669-672, Hepatitis B core (HBc) particles have been extensively exploited as carriers for foreign immunological epitopes in the development of multicomponent vaccines and diagnostic reagents. Crystals of the T = 4 HBc particle were grown in PEG 20 000, ammonium sulfate and various types of alcohols. A temperature jump from 277 or 283 to 290 K was found to enhance crystal growth. A crystal grown using MPD as a cryoprotectant diffracted X-rays to 7.7 Å resolution and data were collected to 99.6% completeness at 8.9 Å. The crystal belongs to space group P212121, with unit-cell parameters a = 352.3, b = 465.5, c = 645.0 Å. The electron-density map reveals a protrusion that is consistent with the N-terminus extending out from the surface of the capsid. The structure presented here supports the idea that N-terminal insertions can be exploited in the development of diagnostic reagents, multicomponent vaccines and delivery vehicles into mammalian cells, We thank Professor K. Murray for providing plasmid pR1-11E. X-ray data collection was supported by BM14UK/ESRF, Grenoble and SRS, Daresbury. WST was supported by a Die NorKen Stiftung Visiting Fellowship and KLH is the recipient of the Darwin Trust Scholarship

Summary Cotranslational protein translocation is a universally conserved process for secretory and membrane protein biosynthesis. Nascent polypeptides emerging from a translating ribosome are either transported across or inserted into the membrane via the ribosome-bound Sec61 channel. Here, we report structures of a mammalian ribosome-Sec61 complex in both idle and translating states, determined to 3.4 and 3.9 Å resolution. The data sets permit building of a near-complete atomic model of the mammalian ribosome, visualization of A/P and P/E hybrid-state tRNAs, and analysis of a nascent polypeptide in the exit tunnel. Unprecedented chemical detail is observed for both the ribosome-Sec61 interaction and the conformational state of Sec61 upon ribosome binding. Comparison of the maps from idle and translating complexes suggests how conformational changes to the Sec61 channel could facilitate translocation of a secreted polypeptide. The high-resolution structure of the mammalian ribosome-Sec61 complex provides a valuable reference for future functional and structural studies., Graphical Abstract, Highlights • A near-complete atomic resolution structure of the mammalian ribosome • Snapshot of a translating ribosome with hybrid state tRNAs and nascent polypeptide • Structures of the Sec61 translocon bound to idle and translating ribosomes • Molecular details of the residues involved in the ribosome-Sec61 interaction, High-resolution structures of the cotranslational translocation complex in two states containing either an idle or actively translating ribosome provide an unprecedented view of the mammalian ribosome, the translocation channel, and molecular details of how they are coupled.