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All eukaryotes utilize a single termination factor, eRF1, to halt translation when the ribosome encounters one of three possible stop codons; here electron cryo-microscopy structures of ribosome–eRF1 complexes in the process of recognizing each stop codon reveal how stop codons are discriminated from sense codons. How mRNA knows when to stop Mammalian messenger RNAs utilize three stop codons, but have a single termination factor, eRF1, that can recognize all three. To understand how eRF1 can distinguish stop codons from sense codons, Alan Brown et al . determined the structures of the mammalian 80S ribosome bound to eRF1 and mRNAs containing each of the stop codons. They find that two nucleotides from the 18S rRNA are stacked with two of the stop codon nucleotides, and the next nucleotide, to compact the mRNA, a conformation that favours stop codons to the exclusion of sense codons. Termination of protein synthesis occurs when a translating ribosome encounters one of three universally conserved stop codons: UAA, UAG or UGA. Release factors recognize stop codons in the ribosomal A-site to mediate release of the nascent chain and recycling of the ribosome. Bacteria decode stop codons using two separate release factors with differing specificities for the second and third bases 1 . By contrast, eukaryotes rely on an evolutionarily unrelated omnipotent release factor (eRF1) to recognize all three stop codons 2 . The molecular basis of eRF1 discrimination for stop codons over sense codons is not known. Here we present cryo-electron microscopy (cryo-EM) structures at 3.5–3.8 Å resolution of mammalian ribosomal complexes containing eRF1 interacting with each of the three stop codons in the A-site. Binding of eRF1 flips nucleotide A1825 of 18S ribosomal RNA so that it stacks on the second and third stop codon bases. This configuration pulls the fourth position base into the A-site, where it is stabilized by stacking against G626 of 18S rRNA. Thus, eRF1 exploits two rRNA nucleotides also used during transfer RNA selection to drive messenger RNA compaction. In this compacted mRNA conformation, stop codons are favoured by a hydrogen-bonding network formed between rRNA and essential eRF1 residues that constrains the identity of the bases. These results provide a molecular framework for eukaryotic stop codon recognition and have implications for future studies on the mechanisms of canonical and premature translation termination 3 , 4 .

The cellular levels and activities of ribosomes directly regulate gene expression during numerous physiological processes. The mechanisms that globally repress translation are incompletely understood. Here, we use electron cryomicroscopy to analyze inactive ribosomes isolated from mammalian reticulocytes, the penultimate stage of red blood cell differentiation. We identify two types of ribosomes that are translationally repressed by protein interactions. The first comprises ribosomes sequestered with elongation factor 2 (eEF2) by SERPINE mRNA binding protein 1 (SERBP1) occupying the ribosomal mRNA entrance channel. The second type are translationally repressed by a novel ribosome-binding protein, interferon-related developmental regulator 2 (IFRD2), which spans the P and E sites and inserts a C-terminal helix into the mRNA exit channel to preclude translation. IFRD2 binds ribosomes with a tRNA occupying a noncanonical binding site, the ‘Z site’, on the ribosome. These structures provide functional insights into how ribosomal interactions may suppress translation to regulate gene expression.

Codanin-1 (CDAN1) is an essential and ubiquitous protein named after congenital dyserythropoietic anemia type I, an autosomal recessive disease that manifests from mutations in CDAN1 or CDIN1 ( CD AN1 i nteracting n uclease 1). CDAN1 interacts with CDIN1 and the paralogous histone H3-H4 chaperones ASF1A ( A nti- S ilencing F unction 1 A) and ASF1B. However, CDAN1 function remains unclear. Here, we analyze CDAN1 complexes using biochemistry, single-particle cryo-EM, and structural predictions. We find that CDAN1 dimerizes and assembles into cytosolic complexes with CDIN1 and multiple copies of ASF1A/B. One CDAN1 can engage two ASF1 through two B-domains commonly found in ASF1 binding partners and two helices that mimic histone H3 binding. We additionally show that ASF1A and ASF1B have different requirements for CDAN1 engagement. Our findings explain how CDAN1 sequesters ASF1A/B by occupying all functional binding sites known to facilitate histone chaperoning and provide molecular-level insights into this enigmatic complex. CDAN1 is an essential protein with unclear function that binds the histone chaperone ASF1. Here, the authors reveal that CDAN1 sequesters multiple copies of ASF1 by engaging all binding sites known to facilitate histone chaperoning.

Tubulins play crucial roles in cell division, intracellular traffic, and cell shape. Tubulin concentration is autoregulated by feedback control of messenger RNA (mRNA) degradation via an unknown mechanism. We identified tetratricopeptide protein 5 (TTC5) as a tubulin-specific ribosome-associating factor that triggers cotranslational degradation of tubulin mRNAs in response to excess soluble tubulin. Structural analysis revealed that TTC5 binds near the ribosome exit tunnel and engages the amino terminus of nascent tubulins. TTC5 mutants incapable of ribosome or nascent tubulin interaction abolished tubulin autoregulation and showed chromosome segregation defects during mitosis. Our findings show how a subset of mRNAs can be targeted for coordinated degradation by a specificity factor that recognizes the nascent polypeptides they encode.

Cells dynamically adjust their protein translation profile to maintain homeostasis in changing environments. During nutrient stress, the kinase general control nonderepressible 2 (GCN2) phosphorylates translation initiation factor eIF2α, initiating the integrated stress response (ISR). To examine the mechanism of GCN2 activation, we have reconstituted this process in vitro, using purified components. We find that recombinant human GCN2 is potently stimulated by ribosomes and, to a lesser extent, by tRNA. Hydrogen/deuterium exchange–mass spectrometry (HDX-MS) mapped GCN2–ribosome interactions to domain II of the uL10 subunit of the ribosomal P-stalk. Using recombinant, purified P-stalk, we showed that this domain of uL10 is the principal component of binding to GCN2; however, the conserved 14-residue C-terminal tails (CTTs) in the P1 and P2 P-stalk proteins are also essential for GCN2 activation. The HisRS-like and kinase domains of GCN2 show conformational changes upon binding recombinant P-stalk complex. Given that the ribosomal P-stalk stimulates the GTPase activity of elongation factors during translation, we propose that the P-stalk could link GCN2 activation to translational stress, leading to initiation of ISR.

Aberrantly stalled ribosomes initiate the ribosome-associated quality control (RQC) and mRNA surveillance pathways for the degradation of potentially toxic peptides and faulty mRNAs. During RQC, ANKZF1 (yeast Vms1p) releases ubiquitinated nascent proteins from 60S ribosomal subunits for proteasomal degradation. Here, we use a cell-free system to show that ANKZF1 and Vms1p sever polypeptidyl-tRNAs on RQC complexes by precisely cleaving off the terminal 3′CCA nucleotides universal to all tRNAs. This produces a tRNA fragment that cannot be aminoacylated until its 3′CCA end is restored. The recycling of ANKZF1-cleaved tRNAs is intact in the mammalian cytosol via a two-step process that requires the removal of a 2′,3′-cyclic phosphate and TRNT1, the sole CCA-adding enzyme that mediates tRNA biogenesis in eukaryotes. TRNT1 also discriminates between properly folded tRNA substrates and aberrant tRNA substrates, selectively tagging the latter for degradation. Thus, ANKZF1 liberates peptidyl-tRNAs from stalled ribosomes such that the tRNA is checked in an obligate way for integrity before reentry into the translation cycle.During ribosome-associated quality control (RQC), ANKZF1 severs polypeptidyl-tRNAs on RQC complexes by cleaving the terminal 3′CCA nucleotides, which leads to tRNA fragments that are ‘quality checked’ and recycled in the cytosol.

Faulty or damaged messenger RNAs are detected by the cell when translating ribosomes stall during elongation and trigger pathways of mRNA decay, nascent protein degradation and ribosome recycling. The most common mRNA defect in eukaryotes is probably inappropriate polyadenylation at near-cognate sites within the coding region. How ribosomes stall selectively when they encounter poly(A) is unclear. Here, we use biochemical and structural approaches in mammalian systems to show that poly-lysine, encoded by poly(A), favors a peptidyl-transfer RNA conformation suboptimal for peptide bond formation. This conformation partially slows elongation, permitting poly(A) mRNA in the ribosome’s decoding center to adopt a ribosomal RNA-stabilized single-stranded helix. The reconfigured decoding center clashes with incoming aminoacyl-tRNA, thereby precluding elongation. Thus, coincidence detection of poly-lysine in the exit tunnel and poly(A) in the decoding center allows ribosomes to detect aberrant mRNAs selectively, stall elongation and trigger downstream quality control pathways essential for cellular homeostasis.

Newly synthesized proteins are triaged between biosynthesis and degradation to maintain cellular homeostasis, but the decision-making mechanisms are unclear. We reconstituted the core reactions for membrane targeting and ubiquitination of nascent tail-anchored membrane proteins to understand how their fate is determined. The central six-component triage system is divided into an uncommitted client-SGTA complex, a self-sufficient targeting module, and an embedded but self-sufficient quality control module. Client-SGTA engagement of the targeting module induces rapid, private, and committed client transfer to TRC40 for successful biosynthesis. Commitment to ubiquitination is dictated primarily by comparatively slower client dissociation from SGTA and nonprivate capture by the BAG6 subunit of the quality control module. Our results provide a paradigm for how priority and time are encoded within a multichaperone triage system.

Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) family proteins are found in all kingdoms of life and in multiple eukaryotic organelles. SPFH proteins assemble into homo- or hetero-oligomeric rings that form domed structures. Most SPFH assemblies also abut a cellular membrane, where they are implicated in diverse functions ranging from membrane organization to protein quality control. However, the precise architectures of different SPFH complexes remain unclear. Here, we report single-particle cryo-EM structures of the endoplasmic reticulum (ER)-resident Erlin1/2 complex and the mitochondrial prohibitin (PHB1/2) complex, revealing assemblies of 13 heterodimers of Erlin1 and Erlin2 and 11 heterodimers of PHB1 and PHB2, respectively. We also describe key interactions underlying the architecture of each complex and conformational heterogeneity of the PHB1/2 complex. Our findings elucidate the distinct stoichiometries and properties of human organellar SPFH complexes and highlight common principles of SPFH complex organization. SPFH proteins oligomerize with distinct stoichiometries in all kingdoms of life. Here, Gao et al. determine cryo-EM structures to elucidate the compositions of the ER-resident Erlin and the mitochondrial prohibitin SPFH complexes implicated in organellar protein quality control.

Rapid and accurate mRNA translation requires efficient codon-dependent delivery of the correct aminoacyl-tRNA (aa-tRNA) to the ribosomal A site. In mammals, this fidelity-determining reaction is facilitated by the GTPase elongation factor-1 alpha (eEF1A), which escorts aa-tRNA as an eEF1A(GTP)-aa-tRNA ternary complex into the ribosome. The structurally unrelated cyclic peptides didemnin B and ternatin-4 bind to the eEF1A(GTP)-aa-tRNA ternary complex and inhibit translation but have different effects on protein synthesis in vitro and in vivo. Here, we employ single-molecule fluorescence imaging and cryogenic electron microscopy to determine how these natural products inhibit translational elongation on mammalian ribosomes. By binding to a common site on eEF1A, didemnin B and ternatin-4 trap eEF1A in an intermediate state of aa-tRNA selection, preventing eEF1A release and aa-tRNA accommodation on the ribosome. We also show that didemnin B and ternatin-4 exhibit distinct effects on the dynamics of aa-tRNA selection that inform on observed disparities in their inhibition efficacies and physiological impacts. These integrated findings underscore the value of dynamics measurements in assessing the mechanism of small-molecule inhibition and highlight potential of single-molecule methods to reveal how distinct natural products differentially impact the human translation mechanism.