Explore the vast range of titles available.

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 .

Gene therapy is a potentially curative medicine for many currently untreatable diseases, and recombinant adeno-associated virus (rAAV) is the most successful gene delivery vehicle for in vivo applications 1 – 3 . However, rAAV-based gene therapy suffers from several limitations, such as constrained DNA cargo size and toxicities caused by non-physiological expression of a transgene 4 – 6 . Here we show that rAAV delivery of a suppressor tRNA (rAAV.sup-tRNA) safely and efficiently rescued a genetic disease in a mouse model carrying a nonsense mutation, and effects lasted for more than 6 months after a single treatment. Mechanistically, this was achieved through a synergistic effect of premature stop codon readthrough and inhibition of nonsense-mediated mRNA decay. rAAV.sup-tRNA had a limited effect on global readthrough at normal stop codons and did not perturb endogenous tRNA homeostasis, as determined by ribosome profiling and tRNA sequencing, respectively. By optimizing the AAV capsid and the route of administration, therapeutic efficacy in various target tissues was achieved, including liver, heart, skeletal muscle and brain. This study demonstrates the feasibility of developing a toolbox of AAV-delivered nonsense suppressor tRNAs operating on premature termination codons (AAV-NoSTOP) to rescue pathogenic nonsense mutations and restore gene function under endogenous regulation. As nonsense mutations account for 11% of pathogenic mutations, AAV-NoSTOP can benefit a large number of patients. AAV-NoSTOP obviates the need to deliver a full-length protein-coding gene that may exceed the rAAV packaging limit, elicit adverse immune responses or cause transgene-related toxicities. It therefore represents a valuable addition to gene therapeutics. The feasibility of adeno-associated-virus-delivered nonsense suppressor tRNAs operating on premature termination codons (AAV-NoSTOP) is explored to restore gene function, using a mouse model of mucopolysaccharidosis type I for proof of concept.

Most amino acids are encoded by multiple codons, and codon choice has strong effects on protein expression. Rare codons are enriched at the N terminus of genes in most organisms, although the causes and effects of this bias are unclear. Here, we measure expression from >14,000 synthetic reporters in Escherichia coli and show that using N-terminal rare codons instead of common ones increases expression by ~14-fold (median 4-fold). We quantify how individual N-terminal codons affect expression and show that these effects shape the sequence of natural genes. Finally, we demonstrate that reduced RNA structure and not codon rarity itself is responsible for expression increases. Our observations resolve controversies over the roles of N-terminal codon bias and suggest a straightforward method for optimizing heterologous gene expression in bacteria.

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. Surprising base pairs fool ribosome 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 pairs 1 . Here we determined the crystal structure at 3.1 Å resolution of the 30S ribosomal subunit in complex with the anticodon stem loop of tRNA Ser 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.

During protein synthesis, nonsense mutations, resulting in premature stop codons (PSCs), produce truncated, inactive protein products. Such defective gene products give rise to many diseases, including cystic fibrosis, Duchenne muscular dystrophy (DMD), and some cancers. Small molecule nonsense suppressors, known as TRIDs (translational read-through–inducing drugs), stimulate stop codon read-through. The best characterized TRIDs are ataluren, which has been approved by the European Medicines Agency for the treatment of DMD, and G418, a structurally dissimilar aminoglycoside. Previously [1], we applied a highly purified in vitro eukaryotic translation system to demonstrate that both aminoglycosides like G418 and more hydrophobic molecules like ataluren stimulate read-through by direct interaction with the cell’s protein synthesis machinery. Our results suggested that they might do so by different mechanisms. Here, we pursue this suggestion through a more-detailed investigation of ataluren and G418 effects on read-through. We find that ataluren stimulation of read-through derives exclusively from its ability to inhibit release factor activity. In contrast, G418 increases functional near-cognate tRNA mispairing with a PSC, resulting from binding to its tight site on the ribosome, with little if any effect on release factor activity. The low toxicity of ataluren suggests that development of new TRIDs exclusively directed toward inhibiting termination should be a priority in combatting PSC diseases. Our results also provide rate measurements of some of the elementary steps during the eukaryotic translation elongation cycle, allowing us to determine how these rates are modified when cognate tRNA is replaced by near-cognate tRNA ± TRIDs.

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.

Premature termination codons (PTCs) account for 10 to 20% of genetic diseases in humans. The gene inactivation resulting from PTCs can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medical applications of this therapeutic strategy. In this study, we developed a reporter cell line and performed high-throughput screening (HTS) to identify potential readthrough inducers. After three successive assays, we isolated 2-guanidinoquinazoline (TLN468). We assessed the clinical potential of this drug as a potent readthrough inducer on the 40 PTCs most frequently responsible for Duchenne muscular dystrophy (DMD). We found that TLN468 was more efficient than gentamicin, and acted on a broader range of sequences, without inducing the readthrough of normal stop codons (TC).

A newly engineered phosphoserine synthetase/tRNA pair allows quantitative insertion of phosphoserine or, when coupled with metabolic rewiring, a non-hydrolyzable analog into protein sequences, leading to high yields of modified constructs for functional analysis. Serine phosphorylation is a key post-translational modification that regulates diverse biological processes. Powerful analytical methods have identified thousands of phosphorylation sites, but many of their functions remain to be deciphered. A key to understanding the function of protein phosphorylation is access to phosphorylated proteins, but this is often challenging or impossible. Here we evolve an orthogonal aminoacyl-tRNA synthetase/tRNA CUA pair that directs the efficient incorporation of phosphoserine (pSer (1)) into recombinant proteins in Escherichia coli . Moreover, combining the orthogonal pair with a metabolically engineered E. coli enables the site-specific incorporation of a nonhydrolyzable analog of pSer. Our approach enables quantitative decoding of the amber stop codon as pSer, and we purify, with yields of several milligrams per liter of culture, proteins bearing biologically relevant phosphorylations that were previously challenging or impossible to access—including phosphorylated ubiquitin and the kinase Nek7, which is synthetically activated by a genetically encoded phosphorylation in its activation loop.

No-go decay (NGD) is one of several messenger RNA (mRNA) surveillance systems dedicated to the removal of defective mRNAs from the available pool. Two interacting factors, Dom34 and Hbs1, are genetically implicated in NGD in yeast. Using a reconstituted yeast translation system, we show that Dom34:Hbs1 interacts with the ribosome to promote subunit dissociation and peptidyl-tRNA drop-off. Our data further indicate that these recycling activities are shared by the homologous translation termination factor complex eRF1:eRF3, suggesting a common ancestral function. Because Dom34:Hbs1 activity exhibits no dependence on either peptide length or A-site codon identity, we propose that this qualitycontrol system functions broadly to recycle ribosomes throughout the translation cycle whenever stalls occur.

Translation termination requires release factors that read a STOP codon in the decoding center and subsequently facilitate the hydrolysis of the nascent peptide chain from the peptidyl tRNA within the ribosome. In human mitochondria eleven open reading frames terminate in the standard UAA or UAG STOP codon, which can be recognized by mtRF1a, the proposed major mitochondrial release factor. However, two transcripts encoding for COX1 and ND6 terminate in the non-conventional AGA or AGG codon, respectively. How translation termination is achieved in these two cases is not known. We address this long-standing open question by showing that the non-canonical release factor mtRF1 is a specialized release factor that triggers COX1 translation termination, while mtRF1a terminates the majority of other mitochondrial translation events including the non-canonical ND6. Loss of mtRF1 leads to isolated COX deficiency and activates the mitochondrial ribosome-associated quality control accompanied by the degradation of COX1 mRNA to prevent an overload of the ribosome rescue system. Taken together, these results establish the role of mtRF1 in mitochondrial translation, which had been a mystery for decades, and lead to a comprehensive picture of translation termination in human mitochondria. How translation termination is achieved for the non-conventional mtDNA-encoded COX1 and ND6 was so far unknown. Here, Nadler et al. address this question by assessing the functions and specificity of the mitochondrial release factors mtRF1 and mtRF1a.