Protein kinase A controls yeast growth in visible light

Background: A wide variety of photosynthetic and non-photosynthetic species sense and respond to light, having developed protective mechanisms to adapt to damaging effects on DNA and proteins. While the biology of UV light-induced damage has been well studied, cellular responses to stress from visible light (400–700 nm) remain poorly understood despite being a regular part of the life cycle of many organisms. Here, we developed a high-throughput method for measuring growth under visible light stress and used it to screen for light sensitivity in the yeast gene deletion collection. Results: We found genes involved in HOG pathway signaling, RNA polymerase II transcription, translation, diphthamide modifications of the translational elongation factor eEF2, and the oxidative stress response to be required for light resistance. Reduced nuclear localization of the transcription factor Msn2 and lower glycogen accumulation indicated higher protein kinase A (cAMP-dependent protein kinase, PKA) activity in many light-sensitive gene deletion strains. We therefore used an ectopic fluorescent PKA reporter and mutants with constitutively altered PKA activity to show that repression of PKA is essential for resistance to visible light. Conclusion: We conclude that yeast photobiology is multifaceted and that protein kinase A plays a key role in the ability of cells to grow upon visible light exposure. We propose that visible light impacts on the biology and evolution of many non-photosynthetic organisms and have practical implications for how organisms are studied in the laboratory, with or without illumination

Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome

Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs. Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2*GTP bound with sordarin. The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics. The IRES rearranges from extended to bent to extended conformations. This inchworm-like movement is coupled with ribosomal inter-subunit rotation and 40S head swivel. eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site. Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center. This unlocks 40S domains, facilitating head swivel and biasing IRES translocation via hitherto-elusive intermediates with PKI captured between the A and P sites. The structures suggest missing links in our understanding of tRNA translocation

RIBOSOME IN THE BALANCE: STRUCTURAL EQUILIBRIUM ENSURES TRANSLATIONAL FIDELITY AND PROPER GENE EXPRESSION

At equilibrium, empty ribosomes freely transit between the rotated and un-rotated states. In translation elongation, the binding of two translation elongation factors to the same general region of the ribosome stabilizes them in one of the two extremes of intersubunit rotation; rotated or unrotated. These stabilized states are resolved by expenditure energy in the form of GTP hydrolysis. Here, mutants of the early assembling integral ribosomal protein uL2 (universal L2) are used to test the generality of this hypothesis. A prior study employing mutants of a late assembling peripheral ribosomal protein suggested that ribosome rotational status determines its affinity for elongation factors, and hence translational fidelity and gene expression. rRNA structure probing analyses reveal that mutations in the uL2 B7b bridge region shift the equilibrium towards the rotated state, propagating rRNA structural changes to all of the functional centers of ribosome. Shift in structural equilibrium affects the biochemical properties of ribosomes: rotated ribosomes favor binding of the eEF2 translocase and disfavor that of the elongation ternary complex. This manifests as specific translational fidelity defects, impacting the expression of genes involved in telomere maintenance. A model is presented here describing how cyclic intersubunit rotation ensures the unidirectionality of translational elongation, and how perturbation of rotational equilibrium affects specific aspects of translational fidelity and cellular gene expression

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

Lipid peroxidation: Production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal

Lipid peroxidation can be described generally as a process under which oxidants such as free radicals attack lipids containing carbon-carbon double bond(s), especially polyunsaturated fatty acids (PUFAs). Over the last four decades, an extensive body of literature regarding lipid peroxidation has shown its important role in cell biology and human health. Since the early 1970s, the total published research articles on the topic of lipid peroxidation was 98 (1970–1974) and has been increasing at almost 135-fold, by up to 13165 in last 4 years (2010–2013). New discoveries about the involvement in cellular physiology and pathology, as well as the control of lipid peroxidation, continue to emerge every day. Given the enormity of this field, this review focuses on biochemical concepts of lipid peroxidation, production, metabolism, and signaling mechanisms of two main omega-6 fatty acids lipid peroxidation products: malondialdehyde (MDA) and, in particular, 4-hydroxy-2-nonenal (4-HNE), summarizing not only its physiological and protective function as signaling molecule stimulating gene expression and cell survival, but also its cytotoxic role inhibiting gene expression and promoting cell death. Finally, overviews of in vivo mammalian model systems used to study the lipid peroxidation process, and common pathological processes linked to MDA and 4-HNE are show