A single amino acid change of translation termination factor eRF1 switches between bipotent and omnipotent stop-codon specificity†

In eukaryotes a single class-1 translation termination factor eRF1 decodes the three stop codons: UAA, UAG and UGA. Some ciliates, like Euplotes, have a variant code, and here eRF1s exhibit UAR-only specificity, whereas UGA is reassigned as a sense codon. Since eukaryote eRF1 stop-codon recognition is associated with its N-terminal domain, structural features should exist in the N domain of ciliate eRF1s that restrict their stop-codon specificity. Using an in vitro reconstituted eukaryotic translation system we demonstrate here that a chimeric eRF1 composed of the N domain of Euplotes aediculatus eRF1 fused to the MC domains of human eRF1 exhibits UAR-only specificity. Functional analysis of eRF1 chimeras constructed by swapping Euplotes N domain sequences with the cognate regions from human eRF1 as well as site-directed mutagenesis of human eRF1 highlighted the crucial role of the alanine residue in position 70 of E. aediculatus eRF1 in restricting UGA decoding. Switching the UAR-only specificity of E. aediculatus eRF1 to omnipotent mode is due to a single point mutation. Furthermore, we examined the influence of eRF3 on the ability of chimeric and mutant eRF1s to induce peptide release in response to different stop codons

Selectivity of stop codon recognition in translation termination is modulated by multiple conformations of GTS loop in eRF1

Translation termination in eukaryotes is catalyzed by two release factors eRF1 and eRF3 in a cooperative manner. The precise mechanism of stop codon discrimination by eRF1 remains obscure, hindering drug development targeting aberrations at translation termination. By solving the solution structures of the wild-type N-domain of human eRF1 exhibited omnipotent specificity, i.e. recognition of all three stop codons, and its unipotent mutant with UGA-only specificity, we found the conserved GTS loop adopting alternate conformations. We propose that structural variability in the GTS loop may underline the switching between omnipotency and unipotency of eRF1, implying the direct access of the GTS loop to the stop codon. To explore such feasibility, we positioned N-domain in a pre-termination ribosomal complex using the binding interface between N-domain and model RNA oligonucleotides mimicking Helix 44 of 18S rRNA. NMR analysis revealed that those duplex RNA containing 2-nt internal loops interact specifically with helix α1 of N-domain, and displace C-domain from a non-covalent complex of N-domain and C-domain, suggesting domain rearrangement in eRF1 that accompanies N-domain accommodation into the ribosomal A site

Genetic Codes with No Dedicated Stop Codon: Context-Dependent Translation Termination

The prevailing view of the nuclear genetic code is that it is largely frozen and unambiguous. Flexibility in the nuclear genetic code has been demonstrated in ciliates that reassign standard stop codons to amino acids, resulting in seven variant genetic codes, including three previously undescribed ones reported here. Surprisingly, in two of these species, we find efficient translation of all 64 codons as standard amino acids and recognition of either one or all three stop codons. How, therefore, does the translation machinery interpret a “stop” codon? We provide evidence, based on ribosomal profiling and “stop” codon depletion shortly before coding sequence ends, that mRNA 3′ ends may contribute to distinguishing stop from sense in a context-dependent manner. We further propose that such context-dependent termination/readthrough suppression near transcript ends enables genetic code evolution

Cell-Free Protein Synthesis

The Nobel Prize in Medicine 1968 for interpretation of the genetic code and its function in protein synthesis and in Chemistry 2009 for studies of the structure and function of the ribosome highlighted the ground-breaking experiment performed on May 15, 1961 by Nirenberg and Matthaei and their principal breakthrough on the creation of "cell-free protein synthesis (CFPS) system". Since then the continuous technical advances have revitalized CFPS system as a simple and powerful technology platform for industrial and high-throughput protein production. CFPS yields exceed grams protein per liter reaction volume and offer several advantages including the ability to easily manipulate the reaction components and conditions to favor protein synthesis, decreased sensitivity to product toxicity, batch reactions last for multiple hours, costs have been reduced orders of magnitude, and suitability for miniaturization and high-throughput applications. With these advantages, there is continuous increasing interest in CFPS system among biotechnologists, molecular biologists and medical or pharmacologists

Fidélité de la traduction chez les eucaryotes. De la molécule au génome

Ce travail porte sur l étude de la fidélité de la traduction chez les eucaryotes d un point de vue mécanistique et génomique. Au cours de ma thèse j'ai développé trois approches :Le premier projet porte sur l étude du rôle du facteur de l élongation eEF2 dans le maintien du cadre de lecture. La stratégie associe une mutagénèse aléatoire du gène EFT2 à un criblage phénotypique, elle permet d isoler des mutants capables d augmenter ou diminuer l efficacité de recodage d une séquence de décalage du cadre de lecture en -1.Le second projet décrit la mise au point d un système de traduction en molécule unique qui permet d étudier le ribosome eucaryote. La traduction est initiée grâce à l IRES CrPV qui a pour caractéristique d être totalement indépendante des facteurs d initiation et de l ARNt initiateur. L élongation de la traduction est détectée grâce au départ d un oligonucléotide fluorescent qui est décroché par l activité hélicase du ribosome. Les résultats de ces expériences constituent une preuve de principe démontrant que l étude de la traduction eucaryote en molécule unique est possible.Le troisième projet est une étude de génomique comparative qui permet de rechercher des événements de recodage ainsi que d autres événements non-conventionnels de la traduction dans le génome de la levure Saccharomyces cerevisiae. L approche est basée sur une recherche d organisations génomiques conservées au sein de 19 génomes de levures. Les gènes candidats sont testés in vivo grâce à un vecteur double rapporteur. Cette étude a permis de mettre en évidence le gène VOA1 qui a été ensuite caractérisé plus en détails.This report describes a study of translation fidelity in Eukarya. Two aspects are tested: the molecular mechanism of recoding and the research of recoding events at the genomic level. During my PhD I have developed three projects:The first project deals with the role of the elongation factor eEF2 in reading frame maintenance. The strategy is based on a random mutagenesis of EFT2 and a phenotypic screening in order to isolate mutants increasing or reducing -1 frameshifting efficiency.The second project describes the development of a single molecule translational system to study the eukaryotic ribosome. Translation initiation is mediated by the CrPV IRES which is initiation factor and initiation tRNA independent. Elongation is monitored with the dissociation of a fluorescent oligonucleotide by the helicase activity of the ribosome. This work is a proof of principle that studying eukaryotic ribosome with single molecule techniques is now feasible.The third project is a comparative genomic approach to search for recoding and unconventional translational events in the genome of yeast Saccharomyces cerevisiae. The approach is based on the detection of conserved genomic organization among 19 Fungi genomes. The candidate genes are then tested in vivo with a dual reporter system. This study allowed the characterization of VOA1 which was further analysed.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Ribosomal frameshifting and transcriptional slippage: From genetic steganography and cryptography to adventitious use.

Genetic decoding is not 'frozen' as was earlier thought, but dynamic. One facet of this is frameshifting that often results in synthesis of a C-terminal region encoded by a new frame. Ribosomal frameshifting is utilized for the synthesis of additional products, for regulatory purposes and for translational 'correction' of problem or 'savior' indels. Utilization for synthesis of additional products occurs prominently in the decoding of mobile chromosomal element and viral genomes. One class of regulatory frameshifting of stable chromosomal genes governs cellular polyamine levels from yeasts to humans. In many cases of productively utilized frameshifting, the proportion of ribosomes that frameshift at a shift-prone site is enhanced by specific nascent peptide or mRNA context features. Such mRNA signals, which can be 5' or 3' of the shift site or both, can act by pairing with ribosomal RNA or as stem loops or pseudoknots even with one component being 4 kb 3' from the shift site. Transcriptional realignment at slippage-prone sequences also generates productively utilized products encoded trans-frame with respect to the genomic sequence. This too can be enhanced by nucleic acid structure. Together with dynamic codon redefinition, frameshifting is one of the forms of recoding that enriches gene expression.This work was supported by grants from Science Foundation Ireland [12/IP/1492 and 13/1A/1853 to J.F.A; 12/IA/1335 to P.V.B.], US. National Institutes of Health [RO3 MH098688 to J.F.A.], the Wellcome Trust [106207 to A.E.F and 094423 to P.V.B.] and the European Research Council (ERC) grant No. 646891 to A.E.F.]This is the final version of the article. It first appeared from Oxford University Press via https://doi.org/10.1093/nar/gkw53

The role of eIF2Bα in the integrity of eIF2B bodies

The eukaryotic initiation factor 2B (eIF2B) is a multisubunit protein composed of two sets of five subunits (α-ε) forming a heterodecamer. eIF2B is a guanine exchange factor (GEF) recycling inactive eukaryotic initiation factor 2 (eIF2) to its active state, being an essential checkpoint for translational control. Following induction of stress, kinases phosphorylate eIF2α, activating the integrated stress response (ISR), subsequently inhibiting eIF2B activity. Mutations within the eIF2Bα-ε subunits can lead to vanishing white matter disease (VWMD) and eIF2Bα variants may cause permanent neonatal diabetes mellitus (PNDM). VWMD and PNDM mutations, primarily affect glial cells and pancreatic β-cells, respectively, suggesting cell type-specific functions of eIF2B. eIF2B bodies are cytoplasmic bodies containing the eIF2B protein. In this study, we highlight bioinformatic tools to optimise the immunocytochemistry detection of eIF2Bα-ε foci. We investigated cell type-specific localisation of eIF2B subunits within neuronal and glial cell types, showing that oligodendrocytes and neuronal cells possess additional eIF2Bα foci, independent of other eIF2B subunits. Additionally, eIF2Bα has been found to be essential for eIF2B foci formation. We have further revealed that eIF2Bɑ spatially interacts with G3BP-containing stress granules (SGs), induced by endoplasmic reticulum (ER) and oxidative stress in a p-eIF2α-dependent manner. EIF2B1L100P/WT mutant cell line shows decreasedformation of SGs following p-eIF2α induction, while SG assembly independent of p-eIF2α was not impacted. Our study showed that in cells depleted of eIF2Bα, large eIF2Bβ-ε foci were not able to form. ISRIB, a molecule known to stabilise the decamer, was able to rescue large foci assembly, establishing a similar functional role to eIF2Bα regarding eIF2B foci formation. Additionally, the assembly of SGs and the localisation of eIF2Bα to these aggregates appeared to play an important role in eIF2B subcomplex formation during ISR activation and the sensing of stress through the p-eIF2α pathway