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Figure S5. VEGF-D 5'UTR mRNA exhibits two structural forms that allow the binding of different proteins

Figure S2. Expression of endogenous VEGF-D in mice tissues

Figure S7. Tumor growth after NSAID treatment

The vascular endothelial growth factor VEGF-D promotes metastasis by inducing lymphangiogenesis and dilatation of the lymphatic vasculature, facilitating tumor cell extravasion. Here we report a novel level of control for VEGF-D expression at the level of protein translation. In human tumor cells, VEGF-D colocalized with eIF4GI and 4E-BP1, which can program increased initiation at IRES motifs on mRNA by the translational initiation complex. In murine tumors, the steady-state level of VEGF-D protein was increased despite the overexpression and dephosphorylation of 4E-BP1, which downregulates protein synthesis, suggesting the presence of an internal ribosome entry site (IRES) in the 5′ UTR of VEGF-D mRNA. We found that nucleolin, a nucleolar protein involved in ribosomal maturation, bound directly to the 5′UTR of VEGF-D mRNA, thereby improving its translation following heat shock stress via IRES activation. Nucleolin blockade by RNAi-mediated silencing or pharmacologic inhibition reduced VEGF-D translation along with a subsequent constriction of lymphatic vessels in tumors. Our results identify nucleolin as a key regulator of VEGF-D expression, deepening understanding of lymphangiogenesis control during tumor formation. Cancer Res; 76(15); 4394–405. ©2016 AACR.

Figure S8. Schematic representation of VEGF-D synthesis in tumor cells

Supplementary figure legend

Figure S6. Polysome profiling of 4T1 cells

Figure S4. Lymphangiogenesis and tumor gowth are not affected by 4T1 or 67NR lentiviral transduction

Figure S3. Podoplanin staining of lymphatic vessels

Escherichia coli CspA is an RNA binding protein that accumulates during cold-shock and stimulates translation of several mRNAs—including its own. Translation in the cold of cspA mRNA involves a cis-acting thermosensor element, which enhances ribosome binding, and the trans-acting action of CspA. Using reconstituted translation systems and probing experiments we show that, at low temperature, CspA specifically promotes the translation of the cspA mRNA folded in the conformation less accessible to the ribosome, which is formed at 37°C but is retained upon cold shock. CspA interacts with its mRNA without inducing large structural rearrangements, but allowing the progression of the ribosomes during the transition from translation initiation to translation elongation. A similar structure-dependent mechanism may be responsible for the CspA-dependent translation stimulation observed with other probed mRNAs, for which the transition to the elongation phase is progressively facilitated during cold acclimation with the accumulation of CspA.

Staphylococcus aureus RsaG is a 3′-untranslated region (3′UTR) derived sRNA from the conserved uhpT gene encoding a glucose-6-phosphate (G6P) transporter expressed in response to extracellular G6P. The transcript uhpT-RsaG undergoes degradation from 5′- to 3′-end by the action of the exoribonucleases J1/J2, which are blocked by a stable hairpin structure at the 5′-end of RsaG, leading to its accumulation. RsaG together with uhpT is induced when bacteria are internalized into host cells or in the presence of mucus-secreting cells. Using MS2-affinity purification coupled with RNA sequencing, several RNAs were identified as targets including mRNAs encoding the transcriptional factors Rex, CcpA, SarA, and the sRNA RsaI. Our data suggested that RsaG contributes to the control of redox homeostasis and adjusts metabolism to changing environmental conditions. RsaG uses different molecular mechanisms to stabilize, degrade, or repress the translation of its mRNA targets. Although RsaG is conserved only in closely related species, the uhpT 3′UTR of the ape pathogen S. simiae harbors an sRNA, whose sequence is highly different, and which does not respond to G6P levels. Our results hypothesized that the 3′UTRs from UhpT transporter encoding mRNAs could have rapidly evolved to enable adaptation to host niches., This work was supported by the Centre National de la Recherche Scientifique (CNRS), by the French National Research Agency ANR (ANR-18-CE12-0025-04 CoNoCo to P.R.). This work of the Interdisciplinary Thematic Institute IMCBio, as part of the ITI 2021–2028 program of the University of Strasbourg, CNRS, and Inserm was supported by IdEx Unistra (ANR-10-IDEX-0002), SFRI-STRAT'US (ANR 20-SFRI-0012), and by EUR IMCBio (IMCBio ANR-17-EURE-0023) under the framework of the French Investments for the Future Program. ED and LB were supported by the “Fondation pour la Recherche Médicale” (FDT201904007957 and ECO202006011534)

SummaryEscherichia coli ribosomal protein S1 is essential for translation initiation of mRNAs and for cellular viability. Two oligonucleotide binding (OB)-fold domains located in the C-terminus of S1 are dispensable for growth, but their deletion causes a cold-shock phenotype, loss of motility and deregulation of RNA mediated stress responses. Surprisingly, the expression of the small regulatory RNA RyhB and one of its repressed target mRNA, sodB, are enhanced in the mutant strain lacking the two OB domains. Using in vivo and in vitro approaches, we show that RyhB retains its capacity to repress translation of target mRNAs in the mutant strain but becomes deficient in triggering rapid turnover of those transcripts. In addition, the mutant is defective in of the final step of the RNase E-dependent maturation of the 16S rRNA. This work unveils an unexpected function of S1 in facilitating ribosome biogenesis and RyhB-dependent mRNA decay mediated by the RNA degradosome. Through its RNA chaperone activity, S1 participates to the coupling between ribosome biogenesis, translation, and RNA decay.

SUMMARYCspA is an RNA binding protein expressed during cold-shock in Escherichia coli, capable of stimulating translation of several mRNAs – including its own – at low temperature. We used reconstituted translation systems to monitor the effects of CspA on the different steps of the translation process and probing experiments to analyze the interactions with its target mRNAs. We specifically focused on cspA mRNA which adopts a cold-induced secondary structure at temperatures below 20°C and a more closed conformation at 37°C. We show that at low temperature CspA specifically promotes the translation of the mRNA folded in the conformation less accessible to the ribosome (37°C form). CspA interacts with its mRNA without inducing large structural rearrangement, does not bind the ribosomal subunits and is not able to stimulate the formation of the translation initiation complexes. On the other hand, CspA promotes the progression of the ribosomes during translation of its mRNA at low temperature and this stimulation is mRNA structure-dependent. A similar structure-dependent mechanism may be responsible for the CspA- dependent translation stimulation observed with other probed mRNAs, for which the transition to the elongation phase is progressively facilitated during cold acclimation with the accumulation of CspA.

Staphylococcus aureus est une bactérie commensale retrouvée chez environ 30 % des individus sains dont elle colonise la peau et la muqueuse nasale. Cependant, c’est également une bactérie pathogène opportuniste responsable d’infections diverses telles que orgelet, ostéomyélite, endocardite, ou encore septicémie en envahissant un grand nombre de tissus et d’organes. Cette bactérie est capable de s’adapter à des conditions hostiles et variées, telles que carence nutritive et stress osmotique, oxydant, ou thermique, ainsi qu’à la réponse immunitaire de l’hôte, car elle produit une grande diversité de facteurs de virulence. La synthèse de ces facteurs est finement régulée par des protéines et des ARN régulateurs majoritairement non codants, souvent désignés par l’abréviation sARN (dérivée de l’anglais, small RNA). Les facteurs de transcription et les systèmes à deux composants contrôlent l’expression des gènes impliqués non seulement dans le métabolisme, mais aussi dans la réponse au stress et la virulence [1]. Par exemple, la protéine du contrôle catabolique (carbon catabolite control protein A, CcpA) a un rôle essentiel dans le choix de la source carbonée en régulant le métabolisme central de la bactérie ainsi que la virulence [2, 3]. CcpA se fixe à une séquence promotrice spécifique appelée cre (catabolite-responsive element), qui est très conservée chez les bactéries à Gram positif [2]. Quant aux sARN, ils interagissent principalement avec leurs ARN messagers (ARNm) cibles. L’hybridation peut conduire à la stabilisation/ déstabilisation de l’ARNm ou à l’activation/répression de sa traduction [4]. Nous avons montré que la transcription du sARN RsaI (RNA Staphylococcus aureus I) est réprimée par CcpA en présence de glucose [5]. L’induction de la synthèse de RsaI signale que la concentration en glucose diminue dans le milieu extracellulaire et que la croissance des bactéries est ralentie. En interagissant avec ses ARNm cibles ou d’autres sARN, il permet à la population bactérienne de modifier son métabolisme lorsque la source carbonée primaire est consommée.

Since its development, single-particle cryogenic electron microscopy (cryo-EM) has played a central role in the study at medium resolution of both bacterial and eukaryotic ribosomal complexes. With the advent of the direct electron detectors and new processing software which allow obtaining structures at atomic resolution, formerly obtained only by X-ray crystallography, cryo-EM has become the method of choice for the structural analysis of the translation machinery. In most of the cases, the ribosomal complexes at different stages of the translation process are assembled in vitro from purified components, which limit the analysis to previously well-characterized complexes with known factors composition. The initiation phase of the protein synthesis is a very dynamic process during which several proteins interact with the translation apparatus leading to the formation of a chronological series of initiation complexes (ICs). Here we describe a method to isolate ICs assembled on natural in vitro transcribed mRNA directly from rabbit reticulocyte lysate (RRL) by sucrose density gradient centrifugation . The Grad-cryo-EM approach allows investigating structures and composition of intermediate ribosomal complexes prepared in near-native condition by cryo-EM and mass spectrometry analyses. This is a powerful approach, which could be used to study translation initiation of any mRNAs, including IRES containing ones, and which could be adapted to different cell extracts.

Since its development, single-particle cryogenic electron microscopy (cryo-EM) has played a central role in the study at medium resolution of both bacterial and eukaryotic ribosomal complexes. With the advent of the direct electron detectors and new processing software which allow obtaining structures at atomic resolution, formerly obtained only by X-ray crystallography, cryo-EM has become the method of choice for the structural analysis of the translation machinery. In most of the cases, the ribosomal complexes at different stages of the translation process are assembled in vitro from purified components, which limit the analysis to previously well-characterized complexes with known factors composition. The initiation phase of the protein synthesis is a very dynamic process during which several proteins interact with the translation apparatus leading to the formation of a chronological series of initiation complexes (ICs). Here we describe a method to isolate ICs assembled on natural in vitro transcribed mRNA directly from rabbit reticulocyte lysate (RRL) by sucrose density gradient centrifugation . The Grad-cryo-EM approach allows investigating structures and composition of intermediate ribosomal complexes prepared in near-native condition by cryo-EM and mass spectrometry analyses. This is a powerful approach, which could be used to study translation initiation of any mRNAs, including IRES containing ones, and which could be adapted to different cell extracts.

Trans‐acting small regulatory RNAs (sRNAs) are key players in the regulation of gene expression in bacteria. There are hundreds of different sRNAs in a typical bacterium, which in contrast to eukaryotic microRNAs are more heterogeneous in length, sequence composition, and secondary structure. The vast majority of sRNAs function post‐transcriptionally by binding to other RNAs (mRNAs, sRNAs) through rather short regions of imperfect sequence complementarity. Besides, every single sRNA may interact with dozens of different target RNAs and impact gene expression either negatively or positively. These facts contributed to the view that the entirety of the regulatory targets of a given sRNA, its targetome, is challenging to identify. However, recent developments show that a more comprehensive sRNAs targetome can be achieved through the combination of experimental and computational approaches. Here, we give a short introduction into these methods followed by a description of two sRNAs, RyhB, and RsaA, to illustrate the particular strengths and weaknesses of these approaches in more details. RyhB is an sRNA involved in iron homeostasis in Enterobacteriaceae, while RsaA is a modulator of virulence in Staphylococcus aureus. Using such a combined strategy, a better appreciation of the sRNA‐dependent regulatory networks is now attainable., The joint application of experimental and the computational tool CopraRNA can provide a comprehensive appreciation of a bacterial sRNA targetome.

The vascular endothelial growth factor VEGF-D promotes metastasis by inducing lymphangiogenesis and dilatation of the lymphatic vasculature, facilitating tumor cell extravasion. Here we report a novel level of control for VEGF-D expression at the level of protein translation. In human tumor cells, VEGF-D colocalized with eIF4GI and 4E-BP1, which can program increased initiation at IRES motifs on mRNA by the translational initiation complex. In murine tumors, the steady-state level of VEGF-D protein was increased despite the overexpression and dephosphorylation of 4E-BP1, which downregulates protein synthesis, suggesting the presence of an internal ribosome entry site (IRES) in the 5′ UTR of VEGF-D mRNA. We found that nucleolin, a nucleolar protein involved in ribosomal maturation, bound directly to the 5′UTR of VEGF-D mRNA, thereby improving its translation following heat shock stress via IRES activation. Nucleolin blockade by RNAi-mediated silencing or pharmacologic inhibition reduced VEGF-D translation along with a subsequent constriction of lymphatic vessels in tumors. Our results identify nucleolin as a key regulator of VEGF-D expression, deepening understanding of lymphangiogenesis control during tumor formation. Cancer Res; 76(15); 4394–405. ©2016 AACR.

Pathogenic bacteria must rapidly adapt to ever‐changing environmental signals resulting in metabolism remodeling. The carbon catabolite repression, mediated by the catabolite control protein A (CcpA), is used to express genes involved in utilization and metabolism of the preferred carbon source. Here, we have identified RsaI as a CcpA‐repressed small non‐coding RNA that is inhibited by high glucose concentrations. When glucose is consumed, RsaI represses translation initiation of mRNAs encoding a permease of glucose uptake and the FN3K enzyme that protects proteins against damage caused by high glucose concentrations. RsaI also binds to the 3′ untranslated region of icaR mRNA encoding the transcriptional repressor of exopolysaccharide production and to sRNAs induced by the uptake of glucose‐6 phosphate or nitric oxide. Furthermore, RsaI expression is accompanied by a decreased transcription of genes involved in carbon catabolism pathway and an activation of genes involved in energy production, fermentation, and nitric oxide detoxification. This multifaceted RNA can be considered as a metabolic signature when glucose becomes scarce and growth is arrested., This work was supported by the Centre National de la Recherche Scientifique (CNRS) to [P.R.] and by the Agence Nationale de la Recherche (ANR, grant ANR‐16‐CE11‐0007‐01, RIBOSTAPH, to [P.R.]). It has also been published under the framework of the LABEX: ANR‐10‐LABX‐0036 NETRNA to [P.R.], a funding from the state managed by the French National Research Agency as part of the investments for the future program. The work is financed by a “Projet international de coopération scientifique” (PICS) No. PICS07507 between France and Spain to [I.C.]. D. Bronesky was supported by Fondation pour la Recherche Médicale (FDT20160435025). A. T‐A is financed by the Spanish Ministry of Economy and Competitiveness (BFU2014‐56698‐P) and the European Research Council Consolidator Grant (646869‐ReguloBac‐3UTR).

Staphylococcus aureus is a Gram-positive major human pathogen involved in a wide range of human infectious diseases (from minor skin infections to septicemia, endocarditis or toxic shock syndrome). The treatment of S. aureus infections is very challenging due to the emergence of multiple antibiotic-resistant isolates. The high diversity of clinical symptoms caused by S. aureus depends on the precise expression of numerous virulence factors and stress response pathways, which are tightly regulated at every level (transcriptional, posttranscriptional, translational, and posttranslational). During the last two decades, it has become evident that small regulatory RNAs (sRNAs) play a major role in fast adaptive responses, mainly by targeting mRNA translation. sRNAs act as antisense RNAs by forming noncontiguous pairings with their target mRNAs and their mechanisms of action vary according to the interaction site. To obtain a global and detailed view of the regulatory networks involved in the adaptive processes of S. aureus, we have adapted the MAPS approach to get individual sRNA targetomes. We also set up different strategies to validate MAPS results and establish sRNA regulatory activities. As this method has been first developed in Gram-negative bacteria, we provide here a protocol for its application in S. aureus and highlight underlying differences. Finally, we discuss several points that have been and could be further improved and provide a workflow file for the automatic analysis of the sequencing in Galaxy.

Pathogenic bacteria must rapidly adapt to ever-changing environmental signals or nutrient availability resulting in metabolism remodeling. The carbon catabolite repression represents a global regulatory system, allowing the bacteria to express genes involved in carbon utilization and metabolization of the preferred carbon source. InStaphylococcus aureus, regulation of catabolite repressing genes is mediated by the carbon catabolite protein A (CcpA). Here, we have identified a CcpA-dependent small non-coding RNA, RsaI that is inhibited by high glucose concentrations. RsaI represses the translation of mRNAs encoding a major permease of glucose uptake, the FN3K enzyme that protects proteins against damages caused by high glucose concentrations, and IcaR, the transcriptional repressor of exopolysaccharide production. Besides, RsaI regulates the activities of other sRNAs responding to the uptake of glucose-6 phosphate or NO. Finally, RsaI inhibits the expression of several enzymes involved in carbon catabolism pathway, and activates genes involved in energy production, fermentation and NO detoxification when the glucose concentration decreases. This multifunctional RNA provides a signature for a metabolic switch when glucose is scarce and growth is arrested.

Pathogenic bacteria must rapidly adapt to ever‐changing environmental signals resulting in metabolism remodeling. The carbon catabolite repression, mediated by the catabolite control protein A (CcpA), is used to express genes involved in utilization and metabolism of the preferred carbon source. Here, we have identified RsaI as a CcpA‐repressed small non‐coding RNA that is inhibited by high glucose concentrations. When glucose is consumed, RsaI represses translation initiation of mRNAs encoding a permease of glucose uptake and the FN3K enzyme that protects proteins against damage caused by high glucose concentrations. RsaI also binds to the 3′ untranslated region of icaR mRNA encoding the transcriptional repressor of exopolysaccharide production and to sRNAs induced by the uptake of glucose‐6 phosphate or nitric oxide. Furthermore, RsaI expression is accompanied by a decreased transcription of genes involved in carbon catabolism pathway and an activation of genes involved in energy production, fermentation, and nitric oxide detoxification. This multifaceted RNA can be considered as a metabolic signature when glucose becomes scarce and growth is arrested.

Chemical probing is often used to gain knowledge on the secondary and tertiary structures of RNA molecules either free or engaged in complexes with ligands. The method monitors the reactivity of each nucleotide towards chemicals of various specificities reflecting the hydrogen bonding environment of each nucleotide within the RNA molecule. In addition, information can be obtained on the binding site of a ligand (noncoding RNAs, protein, metabolites), and on RNA conformational changes that accompanied ligand binding or perturbation of the environmental cues. The detection of the modifications can be obtained either by using end-labeled RNA molecules or by primer extension using reverse transcriptase. The goal of this chapter is to provide the reader with an experimental guide to probe the structure of RNA in vitro and in vivo with the most suitable chemical probes.

Summary Small non-coding RNA molecules (sRNA) are key regulators participating in complex networks, which adapt metabolism in response to environmental changes. In this issue of Molecular Microbiology, and in a related paper in Proc. Natl. Acad. Sci. USA, Moreno et al. (2011) and Sonnleitner et al. (2009) report on novel sRNAs, which act as decoys to inhibit the activity of the master post-transcriptional regulatory protein Crc. Crc is a key protein involved in carbon catabolite repression that optimizes metabolism improving the adaptation of the bacteria to their diverse habitats. Crc is a novel RNA-binding protein that regulates translation of multiple target mRNAs. Two regulatory sRNAs in Pseudomonas putida mimic the natural mRNA targets of Crc and counteract the action of Crc by sequestrating the protein when catabolite repression is absent. Crc trapping by a sRNA is a mechanism reminiscent to the regulation of the repressor of secondary metabolites (RsmA) in Pseudomonas, and highlights the suitability of RNA-dependent regulation to rapidly adjust cell growth in response to environmental changes.

Le prix Nobel de Chimie 2009 a ete decerne a Venkatraman Ramakrishnan (MRC Laboratory of Molecular Biology , Cambridge, Royaume- Uni), Thomas A. Steitz (Howard Hughes Medical Institute , Yale University, Etats-Unis) et Ada E. Yonath (Weizmann Institute of Science , Israel) pour avoir resolu par cristallographie aux rayons X la structure des deux sous-unites (30S et 50S) ainsi que l’assemblage complet du ribosome bacterien. La determination de ces structures, defi inimaginable il y a quelques annees, donne une base architecturale precise a tous les mecanismes d’action de cette nanomachine universelle et complexe qui synthetise les proteines essentielles a la vie de tout organisme. Ces structures offrent un nouvel espoir pour la recherche de nouveaux antibiotiques qui inhiberaient specifiquement le ribosome bacterien, et traiteraient les infections dues a des bacteries qui developpent de plus en plus de resistance aux antibiotiques usuels.

International audience; Translation initiation, the rate-limiting step of the universal process of protein synthesis, proceeds through sequential, tightly regulated steps. In bacteria, the correct messenger RNA start site and the reading frame are selected when, with the help of initiation factors IF1, IF2 and IF3, the initiation codon is decoded in the peptidyl site of the 30S ribosomal subunit by the fMet-tRNA(fMet) anticodon. This yields a 30S initiation complex (30SIC) that is an intermediate in the formation of the 70S initiation complex (70SIC) that occurs on joining of the 50S ribosomal subunit to the 30SIC and release of the initiation factors. The localization of IF2 in the 30SIC has proved to be difficult so far using biochemical approaches, but could now be addressed using cryo-electron microscopy and advanced particle separation techniques on the basis of three-dimensional statistical analysis. Here we report the direct visualization of a 30SIC containing mRNA, fMet-tRNA(fMet) and initiation factors IF1 and GTP-bound IF2. We demonstrate that the fMet-tRNA(fMet) is held in a characteristic and precise position and conformation by two interactions that contribute to the formation of a stable complex: one involves the transfer RNA decoding stem which is buried in the 30S peptidyl site, and the other occurs between the carboxy-terminal domain of IF2 and the tRNA acceptor end. The structure provides insights into the mechanism of 70SIC assembly and rationalizes the rapid activation of GTP hydrolysis triggered on 30SIC-50S joining by showing that the GTP-binding domain of IF2 would directly face the GTPase-activated centre of the 50S subunit.

The protein IF2/eIF5B is one of the few translation initiation factors shared by all three primary domains of life (bacteria, archaea, eukarya). Despite its phylogenetic conservation, the factor is known to present marked functional divergences in the bacteria and the eukarya. In this work, the function in translation of the archaeal homologue (aIF2/5B) has been analysed in detail for the first time using a variety of in vitro assays. The results revealed that the protein is a ribosome-dependent GTPase which strongly stimulates the binding of initiator tRNA to the ribosomes even in the absence of other factors. In agreement with this finding, aIF2/5B enhances the translation of both leadered and leaderless mRNAs when expressed in a cell-free protein-synthesizing system. Moreover, the degree of functional conservation of the IF2-like factors in the archaeal and bacterial lineages was investigated by analysing the behaviour of ‘chimeric’ proteins produced by swapping domains between the Sulfolobus solfataricus aIF2/5B factor and the IF2 protein of the thermophilic bacterium Bacillus stearothermophilus. Beside evidencing similarities and differences between the archaeal and bacterial factors, these experiments have provided insight into the common role played by the IF2/5B proteins in all extant cells.

Bacterial translation initiation factor IF2 was localized on the ribosome by rRNA cleavage using free Cu(II):1,10-orthophenanthroline. The results indicated proximity of IF2 to helix 89, to the sarcin-ricin loop and to helices 43 and 44, which constitute the "L11/thiostrepton" stem-loops of 23S rRNA. These findings prompted an investigation of the L11 contribution to IF2 activity and a re-examination of the controversial issue of the effect on IF2 functions of thiostrepton, a peptide antibiotic known primarily as a powerful inhibitor of translocation. Ribosomes lacking L11 were found to have wild-type capacity to bind IF2 but a strongly reduced ability to elicit its GTPase activity. We found that thiostrepton caused a faster recycling of this factor on and off the 70S ribosomes and 50S subunits, which in turn resulted in an increased rate of the multiple turnover IF2-dependent GTPase. Although thiostrepton did not inhibit the P-site binding of fMet-tRNA, the A-site binding of the EF-Tu-GTP-Phe-tRNA or the activity of the ribosomal peptidyl transferase center (as measured by the formation of fMet-puromycin), it severely inhibited IF2-dependent initiation dipeptide formation. This inhibition can probably be traced back to a thiostrepton-induced distortion of the ribosomal-binding site of IF2, which leads to a non-productive interaction between the ribosome and the aminoacyl-tRNA substrates of the peptidyl transferase reaction. Overall, our data indicate that the translation initiation function of IF2 is as sensitive as the translocation function of EF-G to thiostrepton inhibition.

Bacterial translation initiation factor IF2 is a GTP-binding protein that catalyzes binding of initiator fMet-tRNA in the ribosomal P site. The topographical localization of IF2 on the ribosomal subunits, a prerequisite for understanding the mechanism of initiation complex formation, has remained elusive. Here, we present a model for the positioning of IF2 in the 70S initiation complex as determined by cleavage of rRNA by the chemical nucleases Cu(II):1,10-orthophenanthroline and Fe(II):EDTA tethered to cysteine residues introduced into IF2. Two specific amino acids in the GII domain of IF2 are in proximity to helices H3, H4, H17, and H18 of 16S rRNA. Furthermore, the junction of the C-1 and C-2 domains is in proximity to H89 and the thiostrepton region of 23S rRNA. The docking is further constrained by the requisite proximity of the C-2 domain with P-site-bound tRNA and by the conserved GI domain of the IF2 with the large subunit’s factor-binding center. Comparison of our present findings with previous data further suggests that the IF2 orientation on the 30S subunit changes during the transition from the 30S to 70S initiation complex.

To adapt their metabolism rapidly and constantly in response to environmental variations, bacteria often target the translation initiation process, during which the ribosome assembles on the mRNA. Here, we review different mechanisms of regulation mediated by cis-acting elements, sRNAs and proteins, showing, when possible, their intimate connection with the translational apparatus. Indeed the ribosome itself could play a direct role in several regulatory mechanisms. Different features of the regulatory signals (sequences, structures and their positions on the mRNA) are contributing to the large variety of regulatory mechanisms. Ribosome heterogeneity, variation of individual cells responses and the spatial and temporal organization of the translation process add more layers of complexity. This hampers to define manageable set of rules for bacterial translation initiation control.

Comparative structural studies of ribosomes from various organisms keep offering exciting insights on how species-specific or environment-related structural features of ribosomes may impact translation specificity and its regulation. Although the importance of such features may be less obvious within more closely related organisms, their existence could account for vital yet species-specific mechanisms of translation regulation that would involve stalling, cell survival and antibiotic resistance. Here, we present the first full 70S ribosome structure from Staphylococcus aureus, a Gram-positive pathogenic bacterium, solved by cryo-electron microscopy. Comparative analysis with other known bacterial ribosomes pinpoints several unique features specific to S. aureus around a conserved core, at both the protein and the RNA levels. Our work provides the structural basis for the many studies aiming at understanding translation regulation in S. aureus and for designing drugs against this often multi-resistant pathogen.

The endoribonuclease III (RNase III) belongs to the enzyme family known to process double-stranded RNAs. Staphylococcus aureus RNase III was shown to regulate, in concert with the quorum sensing induced RNAIII, the degradation of several mRNAs encoding virulence factors and the transcriptional repressor of toxins Rot. Two of the mRNA-RNAIII complexes involve fully base paired loop-loop interactions with similar sequences that are cleaved by RNase III at a unique position. We show here that the sequence of the base pairs within the loop-loop interaction is not critical for RNase III cleavage, but that the co-axial stacking of three consecutive helices provides an ideal topology for RNase III recognition. In contrast, RNase III induces several strong cleavages in a regular helix, which carries a sequence similar to the loop-loop interaction. The introduction of a bulged loop that interrupts the regular helix restrains the number of cleavages. This work shows that S. aureus RNase III is able to bind and cleave a variety of RNA-mRNA substrates, and that specific structure elements direct the action of RNase III.

International audience; Crc is a key global translational regulator in Pseudomonads that orchestrates the hierarchy of induction of several catabolic pathways for amino acids, sugars, hydrocarbons or aromatic compounds. In the presence of amino acids, which are preferred carbon sources, Crc inhibits translation of the Pseudomonas putida alkS and benR mRNAs, which code for transcriptional regulators of genes required to assimilate alkanes (hydrocarbons) and benzoate (an aromatic compound), respectively. Crc binds to the 5'-end of these mRNAs, but the sequence and/or structure recognized, and the way in which it inhibits translation, were unknown. We have determined the secondary structure of the alkS mRNA 5'-end through its sensitivity to several ribonucleases and chemical reagents. Footprinting and band-shift assays using variant alkS mRNAs have shown that Crc specifically binds to a short unpaired A-rich sequence located adjacent to the alkS AUG start codon. This interaction is stable enough to prevent formation of the translational initiation complex. A similar Crc-binding site was localized at benR mRNA, upstream of the Shine-Dalgarno sequence. This allowed predicting binding sites at other Crc-regulated genes, deriving a consensus sequence that will help to validate new Crc targets and to discriminate between direct and indirect effects of this regulator.

International audience; Translation initiation is the rate-limiting and most complexly regulated step of protein synthesis in prokaryotes and eukaryotes. In the last few years, cryo-electron microscopy has provided several novel insights into the universal process of translation initiation. Structures of prokaryotic 30S and 70S ribosomal initiation complexes with initiator transfer RNA (tRNA), messenger RNA (mRNA), and initiation factors have recently revealed the mechanism of initiator tRNA recruitment to the assembling ribosomal machinery, involving molecular rearrangements of the ribosome and associated factors. First three-dimensional pictures of the particularly complex eukaryotic translation initiation machinery have been obtained, revealing how initiation factors tune the ribosome for recruiting the mRNA. A comparison of the available prokaryotic and eukaryotic structures shows that--besides significant differences--some key ribosomal features are universally conserved.

Toeprinting was developed to study the formation of ribosomal initiation complexes in bacteria. This approach, based on the inhibition of reverse transcriptase elongation, was used to monitor the effect of ribosomal components and translational factors on the formation of the active ribosomal initiation complex. Moreover, this method offers an easy way to study in vitro how mRNA conformational changes alter ribosome binding at the initiation site. These changes can be induced either by environmental cues (temperature, ion concentration), or by the binding of metabolites, regulatory proteins, and trans-acting RNAs. An experimental guide is given to follow the different steps of the formation of ribosomal initiation complexes in Escherichia coli and Staphylococcus aureus, and to monitor the mechanism of action of several regulators on translation initiation in vitro. Protocols to prepare the ribosome and the subunits are also given for Thermus thermophilus, Staphylococcus aureus, and Escherichia coli.

IF3 has a fidelity function in the initiation of translation, inducing the dissociation of fMet-tRNA(fMet) from the 30 S initiation complexes (30SIC) containing a non-canonical initiation triplet (e.g. AUU) in place of a canonical initiation triplet (e.g., AUG). IF2 has a complementary role, selectively promoting initiator tRNA binding to the ribosome. Here, we used parallel rapid kinetics measurements of GTP hydrolysis, Pi release, light-scattering, and changes in intensities of fluorophore-labeled IF2 and fMet-tRNA(fMet) to determine the effects on both 30SIC formation and 30SIC conversion to 70 S initiation complexes (70SIC) of (a) substituting AUG with AUU, and/or (b) omitting IF3, and/or (c) replacing GTP with the non-hydrolyzable analog GDPCP. We demonstrate that the presence or absence of IF3 has, at most, minor effects on the rate of 30SIC formation using either AUG or AUU as the initiation codon, and conclude that the high affinity of IF2 for both 30 S subunit and initiator tRNA overrides any perturbation of the codon-anticodon interaction resulting from AUU for AUG substitution. In contrast, replacement of AUG by AUU leads to a dramatic reduction in the rate of 70SIC formation from 30SIC upon addition of 50 S subunits. Interpreting our results in the framework of a quantitative kinetic scheme leads to the conclusion that, within the overall process of 70SIC formation, the step most affected by substituting AUU for AUG involves the conversion of an initially labile 70 S ribosome into a more stable complex. In the absence of IF3, the difference between AUG and AUU largely disappears, with each initiation codon affording rapid 70SIC formation, leading to the hypothesis that it is the rate of IF3 dissociation from the 70 S ribosome during IC70S formation that is critical to its fidelity function.