In bacteria, trans-translation is the main rescue system, freeing ribosomes stalled on defective messenger RNAs. This mechanism is driven by small protein B (SmpB) and transfer-messenger RNA (tmRNA), a hybrid RNA known to have both a tRNA-like and an mRNA-like domain. Here we present four cryo-EM structures of the ribosome during trans-translation at resolutions from 3.0 to 3.4 Å. These include the high-resolution structure of the whole pre-accommodated state, as well as structures of the accommodated state, the translocated state, and a translocation intermediate. Together, they shed light on the movements of the tmRNA-SmpB complex in the ribosome, from its delivery by the elongation factor EF-Tu to its passage through the ribosomal A and P sites after the opening of the B1 bridges. Additionally, we describe the interactions between the tmRNA-SmpB complex and the ribosome. These explain why the process does not interfere with canonical translation.

Toxins of toxin-antitoxin systems use diverse mechanisms to control bacterial growth. Here, we focus on the deleterious toxin of the atypical tripartite toxin-antitoxin-chaperone (TAC) system of Mycobacterium tuberculosis, whose inhibition requires the concerted action of the antitoxin and its dedicated SecB-like chaperone. We show that the TAC toxin is a bona fide ribonuclease and identify exact cleavage sites in mRNA targets on a transcriptome-wide scale in vivo. mRNA cleavage by the toxin occurs after the second nucleotide of the ribosomal A-site codon during translation, with a strong preference for CCA codons in vivo. Finally, we report the cryo-EM structure of the ribosome-bound TAC toxin in the presence of native M. tuberculosis cspA mRNA, revealing the specific mechanism by which the TAC toxin interacts with the ribosome and the tRNA in the P-site to cleave its mRNA target.

Because of the ever-increasing multidrug resistance in microorganisms, it is crucial that we find and develop new antibiotics, especially molecules with different targets and mechanisms of action than those of the antibiotics in use today. Translation is a fundamental process that uses a large portion of the cell’s energy, and the ribosome is already the target of more than half of the antibiotics in clinical use. However, this process is highly regulated, and its quality control machinery is actively studied as a possible target for new inhibitors. In bacteria, ribosomal stalling is a frequent event that jeopardizes bacterial wellness, and the most severe form occurs when ribosomes stall at the 3′-end of mRNA molecules devoid of a stop codon. Trans-translation is the principal and most sophisticated quality control mechanism for solving this problem, which would otherwise result in inefficient or even toxic protein synthesis. It is based on the complex made by tmRNA and SmpB, and because trans-translation is absent in eukaryotes, but necessary for bacterial fitness or survival, it is an exciting and realistic target for new antibiotics. Here, we describe the current and future prospects for developing what we hope will be a novel generation of trans-translation inhibitors.

The third step of the catabolism of galactose in mammals is catalyzed by the enzyme galactose-1-phosphate uridylyltransferase (GALT), a homodimeric enzyme with two active sites located in the proximity of the intersubunit interface. Mutations of this enzyme are associated to the rare inborn error of metabolism known as classic galactosemia; in particular, the most common mutation, associated with the most severe phenotype, is the one that replaces Gln188 in the active site of the enzyme with Arg (p.Gln188Arg). In the past, and more recently, the structural effects of this mutation were deduced on the static structure of the wild-type human enzyme; however, we feel that a dynamic view of the proteins is necessary to deeply understand their behavior and obtain tips for possible therapeutic interventions. Thus, we performed molecular dynamics simulations of both wild-type and p.Gln188Arg GALT proteins in the absence or in the presence of the substrates in different conditions of temperature. Our results suggest the importance of the intersubunit interactions for a correct activity of this enzyme and can be used as a starting point for the search of drugs able to rescue the activity of this enzyme in galactosemic patients.

Classic galactosemia is an inborn error of metabolism associated with mutations that impair the activity and the stability of galactose-1-phosphate uridylyltransferase (GALT), catalyzing the third step in galactose metabolism. To date, no treatments (including dietary galactose deprivation) are able to prevent or alleviate the long-term complications affecting galactosemic patients. Evidence that arginine is able to improve the activity of the human enzyme expressed in a prokaryotic model of classic galactosemia has induced researchers to suppose that this amino acid could act as a pharmacochaperone, but no effects were detected in four galactosemic patients treated with this amino acid. Given that no molecular characterizations of the possible effects of arginine on GALT have been performed, and given that the samples of patients treated with arginine are extremely limited for drawing definitive conclusions at the clinical level, we performed computational simulations in order to predict the interactions (if any) between this amino acid and the enzyme. Our results do not support the possibility that arginine could function as a pharmacochaperone for GALT, but information obtained by this study could be useful for identifying, in the future, possible pharmacochaperones for this enzyme.

Caractérisation structurale du mécanisme de sauvetage de la synthèse des protéines chez les bactéries: Le présent travail effectué au cours de cette thèse se concentre sur le mécanisme le plus important impliqué dans le contrôle de la qualité de la synthèse des protéines bactériennes, appelé trans-traduction. La trans- traduction est essentielle pour la survie des cellules bactériennes car elle permet le sauvetage des ribosomes bloqués sur l'ARNm non-stop pendant la traduction, permettant leur recyclage avec la dégradation simultanée du messager défectueux et la dégradation de la chaîne polypeptidique naissante non fonctionnelle. Les deux principaux acteurs impliqués dans ce processus sont l'ARN transfert-messager (ARNtm), une molécule d'ARN chimérique capable de jouer à la fois le rôle d'un ARNt et d'un ARNm, et son partenaire la petite protéine basique B (SmpB), qui est essentielle pour la liaison au ribosome, le positionnement correct du codon de reprise et l'expulsion de l'ARNm non-stop du ribosome. Dans a première section de ce manuscrit, nous expliquons l'étude structurale, par cryo-EM, du processus de trans-traduction qui nous a permis de reconstruire quatre structures atomiques à haute résolution du ribosome dans quatre différentes étapes consécutives de ce mécanisme (pré- accommodation, accommodation, translocation et post-translocation). De cette façon, nous avons pu mettre en évidence les interactions moléculaires au niveau atomique qui sont essentielles à l'ensemble du processus et qui sont particulièrement intéressantes car elles peuvent être utilisées comme cibles contre lesquelles de nouvelles molécules à activité antibactérienne peuvent être développées. Dans la deuxième partie du travail, nous avons concentré notre attention sur la caractérisation structurelle d'un troisième composant impliqué dans la trans-traduction, connu sous le nom de RNase R. La RNase R est une ribonucléase essentielle pour la dégradation de l'ARNm non-stop. Il a été observé qu'elle entre en compétition avec l'ARNtm pour le même site de liaison sur le ribosome, et qu'elle est très dynamique lorsqu'elle interagit avec le ribosome lui-même. Pour cette raison, l'étude a été étendue à un système plus complexe dans lequel nous avons cherché à comprendre le rôle exact des trois acteurs (ARNtm, SmpB et RNase R) lors du décrochage de deux ribosomes sur le même ARNm défectueux (disomes). Cette étude est toujours en cours. Dans le dernier chapitre, nous avons décrit une nouvelle conformation particulière pour l'une des protéines les plus importantes du ribosome, connue sous le nom de bS1. Cette protéine est essentielle dans les premières étapes de la traduction. Dans ce cas, l'un de ses six domaines, normalement connu pour être impliqué dans l'interaction avec le ribosome, présente une conformation particulière qui lui permet d'interagir avec la portion Shine-Dalgarno de l'ARNm, stabilisant ainsi sa liaison avec son homologue à l'extrémité 3' de l'ARNr 16S. Cette observation pourrait ouvrir la porte à une meilleure compréhension du mécanisme d'action de cette protéine.