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Cytosolic long double-stranded RNA (dsRNA), among the most potent proinflammatory signals, is recognized by MDA5. MDA5 binds dsRNA cooperatively, forming helical filaments. ATP hydrolysis by MDA5 fulfills a proofreading function by promoting dissociation of shorter endogenous dsRΝΑs from MDA5 while allowing longer viral dsRNAs to remain bound leading to activation of interferon-β responses. Here, we show that adjacent MDA5 subunits in MDA5-dsRNA filaments hydrolyze ATP cooperatively, inducing cooperative filament disassembly. This amplifies the RNA footprint expansion that accompanies each round of ATP hydrolysis and allows MDA5 to displace tightly bound proteins from dsRNA. Our electron microscopy and biochemical assays show that LGP2 binds to dsRNA at internal binding sites through noncooperative ATP hydrolysis. Unlike MDA5, LGP2 has low nucleic acid selectivity and can hydrolyze GTP and CTP as well as ATP. Binding of LGP2 to dsRNA promotes nucleation of MDA5 filament assembly resulting in shorter filaments. Molecular modeling of the MDA5-LGP2 interface suggests that MDA5 interacts with dsRNA stem-bound rather than end-bound LGP2. We conclude that NTPase-dependent binding of LGP2 to internal sites on dsRNA increases the number and signaling output of MDA5-dsRNA complexes. Our work identifies novel molecular mechanisms contributing the selectivity and sensitivity of cytosolic dsRNA sensing.

Resistance to antimicrobial drugs used to treat bacterial, viral, fungal and parasitic infections is a major health concern requiring a coordinated response across the globe. An important aspect in the fight against antimicrobial resistance is the development of novel drugs that are effective against resistant pathogens. Drug development is a complex trans-disciplinary endeavor, in which structural biology plays a major role by providing detailed functional and mechanistic information on an antimicrobial target and its interactions with small molecule inhibitors. Although X-ray crystallography and nuclear magnetic resonance have until now been the methods of choice to characterize microbial targets and drive structure-based drug development, cryo-electron microscopy is rapidly gaining ground in these areas. In this perspective, we will discuss how cryo-electron microscopy is changing our understanding of an established antimicrobial target, the ribosome, and how methodological developments could help this technique become an integral part of the antimicrobial drug discovery pipeline.

Free L-tryptophan (L-Trp) induces the expression of the Escherichia coli tryptophanase operon, leading to the production of indole from L-Trp. Tryptophanase operon expression is controlled via a mechanism involving the tryptophan-dependent stalling of ribosomes engaged in translation of tnaC, a leader sequence upstream of tnaA that encodes a 24-residue peptide functioning as a sensor for L-Trp. Although extensive biochemical characterization has revealed the elements of the TnaC peptide and the ribosome that are responsible for translational arrest, the molecular mechanism underlying the recognition and response to L-Trp by the TnaC-ribosome complex remains unknown. Here, we use a combined biochemical and structural approach to characterize a variant of TnaC (R23F) in which stalling by L-Trp is enhanced because of reduced cleavage of TnaC(R23F)-peptidyl-tRNA. In contrast to previous data originated from lower resolution structural studies, we show that the TnaC–ribosome complex captures a single L-Trp molecule to undergo tryptophan-dependent termination arrest and that nascent TnaC prevents the catalytic GGQ loop of release factor 2 from adopting an active conformation at the peptidyl transferase center. In addition, we show that the conformation of the L-Trp binding site is not altered by the R23F mutation. This leads us to propose a model in which rates of TnaC-peptidyl-tRNA cleavage by release factor and binding of the L-Trp ligand to the translating ribosome determine the tryptophan sensitivity of the wild-type and mutant TnaC variants. Thus, our study reveals a strategy whereby a nascent peptide assists the bacterial ribosome in sensing a small metabolite.

Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoimmune disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more tightly to synthetic Alu:Alu dsRNA. CryoEM structures MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 side-chain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle-RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via a novel allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides new insights on how MDA5 recognizes dsRNA in health and disease.

Polyamines are essential metabolites that play an important role in cell growth, stress adaptation, and microbial virulence1–3. In order to survive and multiply within a human host, pathogenic bacteria adjust the expression and activity of polyamine biosynthetic enzymes in response to different environmental stresses and metabolic cues2. Here, we show that ornithine capture by the ribosome and the nascent peptide SpeFL controls polyamine synthesis in γ-proteobacteria by inducing the expression of the ornithine decarboxylase SpeF4, via a mechanism involving ribosome stalling and transcription antitermination. In addition, we present the cryo-EM structure of an Escherichia coli (E. coli) ribosome stalled during translation of speFL in the presence of ornithine. The structure shows how the ribosome and the SpeFL sensor domain form a highly selective binding pocket that accommodates a single ornithine molecule but excludes near-cognate ligands. Ornithine pre-associates with the ribosome and is then held in place by the sensor domain, leading to the compaction of the SpeFL effector domain and blocking the action of release factor RF1. Thus, our study not only reveals basic strategies by which nascent peptides assist the ribosome in detecting a specific metabolite, but also provides a framework for assessing how ornithine promotes virulence in several human pathogens.

Bacteria need to rapidly adapt to the changing environment by adjusting their gene expression patterns and enzymatic activities. In most cases, the variations in their habitat involve small molecules that bacteria are able to sense and respond to. The ribosome, the machinery of the cell that catalyzes peptide bond formation, is able to detect metabolites or antibiotics to regulate gene expression via nascent-chain mediated translational arrest. In this mechanism, the peptide that is being translated (arrest peptide) stalls the ribosome by interacting with the walls of the ribosomal tunnel, the cavity through which it reaches the cytoplasm. The arrest may depend solely on the sequence of the peptide or need a small molecule to be triggered. Ribosomal stalling in turn, controls the expression of a gene that is located downstream on the same mRNA. Despite previous biochemical and structural studies, the exact mechanism of sensing of small metabolites by the nascent chain is still unknown. My PhD work focused on: (1) understanding how small molecules are sensed by ribosomal arrest peptides, and (2) a special case of ligand-dependent translational arrest: drug sensing by short arrest peptides.To address the first issue, I studied biochemically and structurally a novel L-ornithine sensing arrest peptide (SpeFL) encoded upstream the speF operon in Escherichia coli. The cryo-EM structure that I solved revealed how a small molecule is sensed by a ribosome nascent chain complex in a highly specific manner. Besides, I showed that the mechanism of induction of the downstream gene speF involves ribosomal arrest at speFL preventing premature Rho-dependent transcriptional termination.On the second part of my thesis, I focused on how a ribosome-targeting antibiotic, erythromycin, is sensed by a short arrest peptide. Erythromycin is able to block translation in a sequence dependent manner, with the (+)X(+) motif being the main stalling motif. Previously published biochemical data suggest that steric and static hindrance caused by the first positively charged amino acid prevents the addition of the second one arresting the ribosome. I solved the cryo-EM structure of a ribosome arrested by an MKFR peptide in the presence of erythromycin that shows otherwise and opens up further investigation on the matter.; Les bactéries doivent s'adapter rapidement aux modifications de leur environnement en ajustant leur modèle d'expression génétique et leurs activités enzymatiques. Dans la plupart des cas, les variations de leur habitat impliquent de petites molécules que les bactéries peuvent détecter et auxquelles elles peuvent réagir. Le ribosome, la machinerie de la cellule qui catalyse la formation de la liaison peptidique, est capable de détecter les métabolites ou les antibiotiques afin de réguler l'expression des gènes, où le peptide naissant au sein du ribosome est capable d’induire l’arrêt de la traduction. Dans ce mécanisme, le peptide en cours de traduction (peptide d'arrêt) bloque le ribosome en interagissant avec les parois du tunnel ribosomal correspondant à la cavité par laquelle le peptide atteint le cytoplasme. L'arrêt peut dépendre uniquement de la séquence du peptide ou bien nécessiter la liaison d’une petite molécule. L’arrêt du ribosome en cours de traduction contrôle à son tour l'expression sur le même ARNm d'un gène situé en aval. Malgré plusieurs études biochimiques et structurales antérieures, le mécanisme exact de détection de ces petits métabolites par le peptide d’arrêt est encore inconnu. Mon travail de doctorat a porté sur : (1) comprendre comment de petites molécules sont détectées par les peptides d'arrêt ribosomaux, et (2) un cas particulier d'arrêt de la traduction dépendant du ligand : la détection des antibiotiques par des peptides d'arrêt courts.Pour répondre au premier problème, j'ai étudié biochimiquement et structurellement un nouveau peptide d'arrêt (appelé SpeFL) qui détecte l’ornithine (un petit métabolite) et qui est codé en amont de l'opéron speF chez Escherichia coli. La structure cryo-EM que j'ai résolue a révélé comment l’ornithine est détectée de manière très spécifique par un complexe ribosomal en cours de traduction. De plus, j'ai montré que le mécanisme d'induction du gène en aval speF implique un arrêt du ribosome au niveau de speFL empêchant ainsi une terminaison prématurée de la transcription Rho-dépendante.Dans la deuxième partie de ma thèse, je me suis concentrée sur la façon dont un antibiotique ciblant les ribosomes, l'érythromycine, est détecté par un peptide d'arrêt court. L'érythromycine est capable de bloquer la traduction de manière séquence-dépendante, où le motif (+)X(+) est le motif principal de blocage. Des données biochimiques publiées antérieurement suggèrent que l'encombrement stérique et électrostatique causé par le premier acide aminé chargé positivement (+) empêche l'addition du second, arrêtant ainsi le ribosome en cours de traduction. La résolution de la structure cryo-EM d'un ribosome arrêté par un peptide MKFR en présence d'érythromycine suggère le contraire, ce qui ouvre la voie à d'autres recherches sur le sujet.

Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoinflammatory disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more stably to synthetic Alu:Alu dsRNA. CryoEM structures of MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 sidechain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle- RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via an allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides insights on how MDA5 recognizes dsRNA in health and disease., Nature Communications, 12 (1), ISSN:2041-1723

Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoinflammatory disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more stably to synthetic Alu:Alu dsRNA. CryoEM structures of MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 sidechain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle- RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via an allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides insights on how MDA5 recognizes dsRNA in health and disease., Human Frontier Science Program

Funder: DH | National Institute for Health Research (NIHR); doi: https://doi.org/10.13039/501100000272, Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoinflammatory disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more stably to synthetic Alu:Alu dsRNA. CryoEM structures of MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 sidechain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle- RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via an allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides insights on how MDA5 recognizes dsRNA in health and disease.

Free L-tryptophan (L-Trp) stalls ribosomes engaged in the synthesis of TnaC, a leader peptide controlling the expression of the Escherichia coli tryptophanase operon. Despite extensive characterization, the molecular mechanism underlying the recognition and response to L-Trp by the TnaC-ribosome complex remains unknown. Here, we use a combined biochemical and structural approach to characterize a TnaC variant (R23F) with greatly enhanced sensitivity for L-Trp. We show that the TnaC–ribosome complex captures a single L-Trp molecule to undergo termination arrest and that nascent TnaC prevents the catalytic GGQ loop of release factor 2 from adopting an active conformation at the peptidyl transferase center. Importantly, the L-Trp binding site is not altered by the R23F mutation, suggesting that the relative rates of L-Trp binding and peptidyl-tRNA cleavage determine the tryptophan sensitivity of each variant. Thus, our study reveals a strategy whereby a nascent peptide assists the ribosome in detecting a small metabolite., Bacteria adjust the expression of some of their metabolic enzymes through metabolite-sensing ribosome nascent chain complexes. Here the authors present a cryo-EM structure of an E. coli ribosome stalled during translation of the TnaC leader peptide and propose a model for L-Trp dependent ribosome stalling where L-Trp competes with release factor 2 for binding to the TnaC-ribosome complex.

Funder: National Institute for Health Research (NIHR), Funder: Medical Research Council, Funder: Biotechnology and Biological Sciences Research Council, Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoinflammatory disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more stably to synthetic Alu:Alu dsRNA. CryoEM structures of MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 sidechain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle- RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via an allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides insights on how MDA5 recognizes dsRNA in health and disease.