Termination of protein synthesis occurs when a translating ribosome encounters one of three universally conserved stop codons: UGA, UAA, or UAG. Release factors recognise 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 bases1. By contrast, eukaryotes rely on an evolutionarily unrelated omnipotent release factor (eRF1) to recognise all three stop codons2. The molecular basis of eRF1 discrimination for stop codons over sense codons is not known. Here, we present electron cryo-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 rRNA 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 stabilised by stacking against G626 of 18S rRNA. Thus, eRF1 exploits two rRNA nucleotides also used during tRNA selection to drive mRNA compaction. Stop codons are favoured in this compacted mRNA conformation by a hydrogen-bonding network with 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 termination3,4.

SummaryIn eukaryotes, accurate protein synthesis relies on a family of translational GTPases that pair with specific decoding factors to decipher the mRNA code on ribosomes. We present structures of the mammalian ribosome engaged with decoding factor⋅GTPase complexes representing intermediates of translation elongation (aminoacyl-tRNA⋅eEF1A), termination (eRF1⋅eRF3), and ribosome rescue (Pelota⋅Hbs1l). Comparative analyses reveal that each decoding factor exploits the plasticity of the ribosomal decoding center to differentially remodel ribosomal proteins and rRNA. This leads to varying degrees of large-scale ribosome movements and implies distinct mechanisms for communicating information from the decoding center to each GTPase. Additional structural snapshots of the translation termination pathway reveal the conformational changes that choreograph the accommodation of decoding factors into the peptidyl transferase center. Our results provide a structural framework for how different states of the mammalian ribosome are selectively recognized by the appropriate decoding factor⋅GTPase complex to ensure translational fidelity.

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.

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.DOI: http://dx.doi.org/10.7554/eLife.13567.001

Epidermal cells from adult guinea pig skin attach and differentiate preferentially on substrates of type IV (basement membrane) collagen, compared to those of types I-III collagen. In contrast, guinea pig dermal fibroblasts attach equally well to all four collagen substrates. Fibronectin mediates the attachment of fibroblasts but not of epidermal cells to collagen. KEY WORDS epidermal cells adhesion collagen -basement membrane differentiationSeveral chemically and genetically distinct collagens are found in mammalian species. Types I and III collagen are widely distributed (14). Type II collagen is found only in cartilage (13) and type IV in basement membranes (9). The matrices that these collagens form differ in appearance and physical characteristics. Collagen substrates enhance the attachment (10), growth (2), and differentiation (5) of various cell types. Such observations suggest that the interaction of cells with collagen may have important functions in vivo. Some clue to the mechanism(s) operative in this interaction came from recent studies (10, 11) which indicate that fibroblasts do not bind directly to collagen substrates but that a membrane protein (the collagen-cen attachment protein) mediates the attachment of cells to collagen substrates. This protein (or proteins) is similar or identical to CSP (23), LETS protein (7) and cold insoluble globulin (15,19), and has been named fibronectin (18). Fetal calf serum used in cell culture is a rich source of fibronectin (10).It seemed conceivable to us that collagen-cell interactions depend not only on mediator proteins but also on either the chemical characteristics of the collagen substrate or on the nature of the attaching cell. To test this hypothesis, we compared the attachment of two functionally diverse cell types, fibroblasts and epidermal cells, to various collagen substrates and their dependence on fibronectin for the mediation of attachment. In contrast to fibroblasts, epidermal cells from postembryonic animals are difficult to culture in vitro unless conditioned media and collagen substrates (8) or feeder layers of fibroblasts (17) are used. We show here that epidermal cells differ from fibroblasts in their attachment to collagen substrates and that epidermal cells attach best to type IV collagen whereas fibroblasts attach equally well to all collagen types. Further, we demonstrate that fibronectin enhances the attachment of fibroblasts to collagen but does not affect epidermal cell attachment. MATERIALS AND METHODS Collagen SubstratesType I collagen was prepared from the skins of lathyritic rats (1), type II from a rat chondrosarcoma (20), type III from fetal calf skin (4), and type IV from a murine sarcoma (16,22). The purity and identity to standards of these collagens was confn'med by sodium dodecyl sulfate (SDS) gel electrophoresis (12) and amino acid analysis. The various collagens (1 mg/ml in 0.5 M acetic acid) were stored at -20"C. To study the attachment of cells, the stock solutions of collagen were diluted to 10 /~g/ml with wate...

Ancient components of the ribosome, inferred from a consensus of previous work, were constructed in silico, in vitro and in vivo. The resulting model of the ancestral ribosome presented here incorporates ∼20% of the extant 23S rRNA and fragments of five ribosomal proteins. We test hypotheses that ancestral rRNA can: (i) assume canonical 23S rRNA-like secondary structure, (ii) assume canonical tertiary structure and (iii) form native complexes with ribosomal protein fragments. Footprinting experiments support formation of predicted secondary and tertiary structure. Gel shift, spectroscopic and yeast three-hybrid assays show specific interactions between ancestral rRNA and ribosomal protein fragments, independent of other, more recent, components of the ribosome. This robustness suggests that the catalytic core of the ribosome is an ancient construct that has survived billions of years of evolution without major changes in structure. Collectively, the data here support a model in which ancestors of the large and small subunits originated and evolved independently of each other, with autonomous functionalities.

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Primary and passaged cultures of fibroblasts (RBMFs) raised from the bone marrow stroma of young rabbits were treated with pulsed electromagnetic fields (PEMFs) from the start of each culture until 1 week after they became confluent. the PEMF treatment had no effect on cell proliferation, estimated by phase contrast microscopy, by 3H-thymidine incorporation into DNA, or by total DNA assay. Collagen production, estimated by conversion of 3H-proline to 3H-hydroxyproline in nondialyzable material was markedly elevated in postconfluent cultures, but not in cultures that had only just reached confluence. About 65 of 3H-hydroxyproline was in low molecular weight form, and a correlation between collagen breakdown and cyclic AMP (cAMP) levels in RBMFs was demonstrated by adding dibutyryl cAMP or prostaglandin E3 (PGE2) to the culture medium concurrently with 3H-proline. The PEMF apparatus caused an insufficient temperature rise (less than 0.1 degree C) to account for these results. We propose that the rise in collagen production is consistent with the hypothesis that PEMFs act by reducing cAMP levels in RBMFs, and that thermal effects are insignificant.