La traduction est un processus itératif réalisé par le ribosome. Chez Escherichia coli, le ribosome est composé d’une grande sous-unité 50S et d’une petite sous-unité 30S (S correspondant au coefficient de sédimentation). La traduction débute par la mise en place d’une interaction entre le codon d’initiation de l’ARNm et l’anticodon de l’ARNt initiateur. Cette interaction, finement régulée par les facteurs d’initiations IF1, IF2 et IF3, conduit à la formation du complexe d’initiation 30S (30SIC). Par titration calorimétrique isotherme (ITC), nous avons disséqué la thermodynamique de l’ensemble des voies possibles de formation du 30SIC. Sur la base des affinités mesurées, il a été possible d’en déduire un ordre d’assemblage préférentiel. Par cryo-microscopie électronique, nous avons ensuite essayé d’obtenir la structure de ce complexe à haute résolution. La fixation du 50S représente la dernière étape de l’initiation. Par ITC, nous avons cherché à en déterminer les paramètres thermodynamiques, puis nous avons poursuivi avec l’élongation en commençant par étudier l’incorporation d’un aminoacyl-ARNt. Enfin, la réalisation d’une étude comparative par ITC de trois antibiotiques capables de se fixer au tunnel de sortie du peptide, nous a permis d’identifier les forces moléculaires mises en jeu lors de leur interaction avec le ribosome. / Translation is an iterative process achieved by the ribosomal machinery. In Escherichia coli, the ribosome is composed of a large 50S subunit and a small 30S subunit (S being the sedimentation coefficient). Translation begins with the establishment of the interaction between the mRNA codon and the initiator tRNA anticodon. This interaction, under the control of initiation factors IF1, IF2 and IF3, leads to the formation of the 30S initiation complex (30SIC). Using isothermal titration calorimetry (ITC), we explored the thermodynamic landscape of all possible pathways for 30SIC formation. Based on affinities derived from ITC, we propose a preferred assembly pathway. Using cryo-electron microscopy, this knowledge was used to obtain high-resolution structures of 30SIC intermediates. Binding of the 50S is the last step for initiation. Using ITC, thermodynamic parameters were derived followed by the incorporation of an aminoacyl-tRNA. Lastly, we realized, using ITC, a comparative study of three antibiotics binding to the nascent peptide exit tunnel of the ribosome. This study leads us to determine the molecular forces involved in their interaction with the ribosome.

Translation initiation is a complex process which results in the assembly of the elongation competent 80S ribosome from the 40S and 60S ribosomal subunits, the initiator tRNA and mRNA, and is orchestrated by numerous eukaryotic initiation factors (eIFs). Although it represents one of the most regulated processes of gene expression, the exact mechanism of one of the key steps of translation initiation - mRNA recruitment to the 43S pre-initiation complex (PIC) - is still only poorly understood. Recent studies indicated that besides eIF4F and poly(A)-binding protein, also eIF3 might play an important, if not crucial, role in this step. In our laboratory, we recently identified a 10 Ala substitution (Box37) in the a/TIF32 subunit of Saccharomyces cerevisiae eIF3, which interfered with translation initiation rates. Detailed analysis showed that this mutation significantly reduces the amounts of model mRNA in the gradient fractions containing 48S PICs as the only detectable effect in vivo. Moreover, a recently solved crystal structure of the N-terminal part of a/TIF32 pointed to two Box37 residues, Arg363 and Lys364, both proposed to contribute to one of the positive, potentially RNA-binding areas on the a/TIF32 surface. The fact that also their substitutions with alanines severely impaired the mRNA recruitment...

The transcription initiation is the first step in gene expression and an important regulation step in all living organisms. In bacteria, it has been proposed that DNA bending and its wrapping on the surface of E. coli RNAP might facilitate the opening of the transcription bubble, which is necessary for the initiation of gene transcription. In this work, it is shown the first structural study to evaluate a DNA wrapping model, including its length and the relative position in the bacterial transcription initiation complex (RP complex), assembled between RNA polymerase-σ70 holoenzyme (RNAP) and a λPR promoter (-100 to +30 wild type). RP complex was prepared and negatively stained with 2% uranyl acetate on a thin-carbon coated grid and the data acquisition of 500 images was performed in a JEM-2100 (JEOL, Japan) microscope equipped with an F-416 CMOS camera (TVIPS, Germany). Single particle analysis of 16,015 particles, grouped in 666 class-averages, was conducted using IMAGIC 4D software (Image Science, Germany) to obtain a three-dimensional model of the RP complex at 20Å resolution. After the rigid-body fitting of the RNAP crystallographic structure (PDB 4YG2) and the modeled DNA promoter, it was observed that the regions 1.2 and 4.2 of the σ70 subunit interacts with the consensus zones, -10 and -35 hexamers of the promoter. Furthermore, it was possible to observe that αCTDs (C-terminal domain) in both alpha subunits would be oriented to facilitate the interaction with the first and second UP-elements regions, respectively (centered around –50 and -75 positions in the promoter). These was enabled by the presence of the characteristics motifs helix-hairpin-helix in these domains. In addition, the downstream DNA, from the transcription bubble, appears to be inside the protein main channel, oriented in a way to enable interactions with the RNAP clamp and jaws. Finally, it was observed that the DNA wrapping has ~32 nm of total length and involves a promoter bent of ~255° around the RNAP surface. The 3D-model obtained in this study is the very first direct structural confirmation of the DNA promoter wrapping in a bacterial transcription initiation complex. / A iniciação da transcrição é o primeiro passo na expressão gênica e importante ponto de regulação em todos os organismos vivos. Em bactérias, foi proposto que o enovelamento do DNA na superfície da RNAP de E. coli pode facilitar a abertura da bolha de transcrição, necessária para o início da transcrição gênica. Neste trabalho, é apresentado o primeiro estudo estrutural direto para avaliar o comprimento do enovelamento do DNA e sua posição no complexo de iniciação da transcrição bacteriana (complexo RP), montado entre a holoenzima RNA polimerase-σ70 (RNAP) e um promotor λPR (-100 para +30, tipo selvagem). Amostras do complexo RP foram preparadas e contrastadas negativamente com 2% de acetato de uranila em uma grade com filme fino de carbono e a aquisição de 500 imagens foi realizada em um microscópio JEM-2100 (Jeol, Japão) equipado com uma câmera CMOS F-416 (TVIPS, Alemanha). A análise de partículas isoladas de 16.015 partículas, agrupadas em 666 médias de classe, foi conduzida usando o software IMAGIC 4D (Image Science, Alemanha) para obter um modelo tridimensional do complexo RP, a 20Å de resolução, estimado pelo critério de ½ bit. Após o ajuste de corpo rígido da estrutura cristalográfica da RNAP (PDB 4YG2) e do promotor de DNA modelado, observou-se que as regiões 1.2 e 4.2 da subunidade σ70 interagem com as zonas de consenso, hexâmeros -10 e -35, do promotor. Além disso, foi possível observar que os αCTDs (domínio C-terminal) em ambas as subunidades alfa estariam orientados para facilitar uma possível interação com a primeira e segundas regiões dos elementos UP, respectivamente (centradas em torno das posições –50 e -75 do promotor). Estas seriam possíveis devido à presença de alguns motivos de características hélice-grampo-hélice nesses domínios. Além disso, a região do promotor, downstream da bolha de transcrição, parece estar dentro do canal principal da proteína, orientado de forma a possibilitar interações com o clamp e jaw da RNAP. Finalmente, foi observado que o comprimento total do enovelamento de DNA envolve cerca de 32 nm e 255° de rotação do DNA ao redor da superfície da RNAP. Portanto, este modelo 3D é a primeira confirmação estrutural direta do enovelamento de DNA em um complexo bacteriano de iniciação da transcrição.

Le complexe SAGA (Spt-Ada-Gcn5 acetyl transferase) est un co-activateur transcriptionel, conservé chez les eucaryotes, qui participent à la transcription d’environ 10% des gènes chez la levure, où il fait le lien entre les composants du complexe de pré-initiation, tel que la TATA-box Binding Protein (TBP) et des activateurs, et modifie les histones dans le contexte de la chromatine (acétylation et déubiquitination). Ces travaux de thèse ont permis de décrire l’architecture moléculaire du complexe observée par microscopie électronique. Nous avons pu (i) localiser le module de déubiquitination au sein du complexe entier et ainsi (ii) définir une zone d’interaction avec le nucléosome ; (iii) montrer la présence de deux sites d’interaction avec la protéine TBP situé au niveau d’une « pince »moléculaire ; (iv) observer un lien fonctionnel entre le module de déubiquitination, en particulier de la protéine Sgf73, et les conformations adoptées par cette pince. / The SAGA complex (Spt-Ada-Gcn5 acetyl transferase) is a transcriptional coactivator, highly conserved in eukaryotes, involved in the transcription of 10% of the genes in yeast, where it bridges the components of the pre-initiation complex such as the TATA-box Binding Protein (TBP) and activators, as well as modifies histones in the chromatin template (acetylation and deubiquitination). This work has revealed the molecular architecture of the complex observed by electron microscopy. We could (i) localize the deubiquitination module within the whole complex and thus (ii) define the interaction surface with the nucleosome; (iii) reveal the presence of two TBP-interacting surfaces localized at the tips of a molecular clamp; (iv) observe a functional link between the deubiquitination module, in particular the Sgf73 protein, and the conformation adopted by this clamp.

Malgré les thérapies anti-cancéreuses ciblées, beaucoup de patients récidivent à cause de la résistance aux traitements qui constitue un problème clinique majeur. Cette résistance est soutenue par la reprogrammation métabolique et traductionnelle. La synthèse des protéines oncogéniques fait appel au complexe d’initiation de la traduction eucaryote 4F (eIF4F), compris des facteurs : eIF4A, eIF4E et eIF4G. Il est ainsi possible de cibler la synthèse protéique spécifique aux cellules cancéreuses par des inhibiteurs de l’initiation de la traduction. Nous proposons que le ciblage de la machinerie traductionnelle, via l’inhibition du eIF4A (eIF4Ai), affecte particulièrement les cellules cancéreuses. Mais est-ce qu’il est en mesure d’atténuer la résistance aux inhibiteurs de kinase (IKs) et d’en empêcher l’adaptation et la reprogrammation métabolique? L’efficacité des eIF4Ais est vérifiée par l’évaluation de la croissance et de la mort cellulaire (Annexine V) dans des cellules de mélanome, sensibles et résistantes aux IKs. Celle-ci est accompagnée de la réduction de la synthèse protéique évaluée par profilage polysomique, de la baisse des cibles de l'eIF4Ai (BCL-2, CDK4...) quantifiées par western-blots, d’un important stress bioénergétique mesuré par Seahorse et du contrôle des principales voies métaboliques analysées par GCMS. L’analyse du profilage polysomique, de l’ARNseq et du métabolome permettront de mettre en évidence les réseaux qui soutiennent l’efficacité des eIF4Ais et les mécanismes moléculaires sous-jacents à l’efficacité de cette thérapie. Ces derniers seront par la suite validés par des approches génétiques évaluant les principaux gènes et voies métaboliques qui y sont impliqués. Cette étude comblera de grosses lacunes dans les connaissances relatives aux mécanismes moléculaires qui soutiennent la résistance aux IKs afin d’améliorer leur efficacité en clinique. / Despite advances in research and development of targeted cancer therapies, many patients relapse due to treatment resistance. This is the case of melanoma resistant to BRAF inhibitors (BRAFi). 40-50% of melanoma cancer cells express the constitutively active oncoprotein BRAFV600E, leading to metabolic and translational reprogramming that is proposed to support resistance to cancer therapies. Studies have shown the importance of the eukaryotic translation initiation complex 4F (eIF4F), including factors: eIF4A, eIF4E and eIF4G, in the oncogenic proteins’ synthesis (e.g. growth factors, metabolic factors, etc.). The cancer cells translatome is distinct from the normal cells translatome. It is thus possible to target protein synthesis specific to cancer cells using molecules that inhibit translation initiation, the limiting phase in this process, affecting the cancer cells specifically. We propose that targeting the translational machinery via eIF4A inhibitors (eIF4Ai) would attenuate resistance to kinase inhibitors (KIs) and we seek to dissect the underlying molecular mechanisms. The efficacy of eIF4Ais in BRAFi sensitive and/or resistant melanoma is evaluated showing that eIF4Ais inhibit cell growth and induce melanoma cells death, using growth curves and FACS (Annexin_V). This is accompanied by a protein synthesis reduction, evaluated by polysome profiling showing a decrease in eIF4Ais treated cells and western-blots showing a decrease in translational targets of eIF4Ai (BCL-2, CDK4, etc.). A significant bioenergetic stress is measured by Seahorse and the control of the main metabolic pathways including glycolysis, TCA cycle and amino acids metabolism is analyzed by GCMS. Then the analysis of the translatome by polysome profiling and RNAseq and of the metabolome by GCMS/LCMS and Seahorse shed light on the translational networks that dictate metabolic reprogramming supporting eIF4Ai efficacy. Finally, the molecular mechanisms underlying the efficacy of eIF4Ai will be identified using genetic approaches to validate the main genes and metabolites and/or corresponding metabolic pathways involved in the response to eIF4Ai of the kinase inhibitor resistant melanoma. This study will fill large gaps in knowledge about the molecular mechanisms that support resistance to KIs in order to improve their clinical efficacy.