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Ribosome biogenesis is a complex and highly accurate conservative process of ribosomal subunit maturation followed by association. Subunit maturation comprises sequential stages of ribosomal RNA and proteins’ folding, modification and binding, with the involvement of numerous RNAses, helicases, GTPases, chaperones, RNA, protein-modifying enzymes, and assembly factors. One such assembly factor involved in bacterial 30S subunit maturation is ribosomal binding factor A (RbfA). In this study, we present the crystal (determined at 2.2 Å resolution) and NMR structures of RbfA as well as the 2.9 Å resolution cryo-EM reconstruction of the 30S–RbfA complex from Staphylococcus aureus (S. aureus). Additionally, we show that the manner of RbfA action on the small ribosomal subunit during its maturation is shared between bacteria and mitochondria. The obtained results clarify the function of RbfA in the 30S maturation process and its role in ribosome functioning in general. Furthermore, given that S. aureus is a serious human pathogen, this study provides an additional prospect to develop antimicrobials targeting bacterial pathogens.

Ribosomal silencing factor S (RsfS) is a conserved protein that plays a role in the mechanisms of ribosome shutdown and cell survival during starvation. Recent studies demonstrated the involvement of RsfS in the biogenesis of the large ribosomal subunit. RsfS binds to the uL14 ribosomal protein on the large ribosomal subunit and prevents its association with the small subunit. Here, we estimated the contribution of RsfS amino acid side chains at the interface between RsfS and uL14 to RsfS anti-association function in

Ribosome-binding factor A (RbfA) from Staphylococcus aureus is a cold adaptation protein that is required for the growth of pathogenic cells at low temperatures (10-15°C). RbfA is involved in the processing of 16S rRNA, as well as in the assembly and stabilization of the small 30S ribosomal subunit. Structural studies of the 30S-RbfA complex will help to better understand their interaction, the mechanism of such complexes, and the fundamental process such as 30S subunit assembly that determines and controls the overall level of protein biosynthesis. This article describes protocols for preparation of RbfA and the small 30S ribosomal subunits and reconstitution and optimization of the 30S-RbfA complex to obtain samples suitable for cryo-electron microscopy studies.

Elongation factor P (EF-P) is a translation protein factor that plays an important role in specialized translation of consecutive proline amino acid motifs. EF-P is an essential protein for cell fitness in native environmental conditions. It regulates synthesis of proteins involved in bacterial motility, environmental adaptation and bacterial virulence, thus making EF-P a potential drug target. In the present study, we determined the solution and crystal structure of EF-P from the pathogenic bacteria Staphylococcus aureus at 1.48 Å resolution. The structure can serve as a platform for structure-based drug design of novel antibiotics to combat the growing antibiotic resistance of S. aureus.

International audience; Staphylococcus aureus is a bacterial pathogen and one of the leading causes of healthcare-acquired infections in the world. The growing antibiotic resistance of S. aureus obliges us to search for new drugs Accepted Article This article is protected by copyright. All rights reserved and treatments. As the majority of antibiotics target the ribosome, knowledge of its detailed structure is crucial for drug development. Here, we report the Cryo-EM reconstruction at 3.2 Å resolution of the S. aureus ribosome with P-site tRNA, messenger RNA and ten RNA modification sites previously not assigned or visualized. The resulting model is the most precise and complete high-resolution structure to date of the S. aureus 70S ribosome with functional ligands.

Antibiotic‐resistant Staphylococcus aureus is becoming a major burden on health care systems in many countries, necessitating the identification of new targets for antibiotic development. Elongation Factor P (EF‐P) is a highly conserved elongation protein factor that plays an important role in protein synthesis and bacteria virulence. EF‐P undergoes unique posttranslational modifications in a stepwise manner to function correctly, but experimental information on EF‐P posttranslational modifications is currently lacking for S. aureus. Here, we expressed EF‐P in S. aureus to analyze its posttranslational modifications by mass spectrometry and report experimental proof of 5‐aminopentanol modification of S. aureus EF‐P., Elongation Factor P (EF‐P) is a conserved protein involved in balance regulation, polyproline motif‐containing proteins, stress resistance and virulence. The interaction between the EF‐P posttranslational modification and the CCA end of the acceptor stem of the initiator tRNA growing peptide allows it to be properly evacuated from the ribosome. We performed mass spectrometry analysis of EF‐P from S. aureus and identified the presence of 5‐aminopentanolation.

Elongation Factor P (EF-P) is a 20.5 kDa protein that provides specialized translation of special stalling amino acid motifs. Proteins with stalling motifs are often involved in various processes, including stress resistance and virulence. Thus it has been shown that the virulent properties of microorganisms can be significantly reduced if the work of EF-P is disrupted. In order to elucidate the structure, dynamics and function of EF-P from Staphylococcus aureus (S. aureus), here we report backbone and side chains 1H, 13C and 15N chemical shift assignments of EF-P. Analysis of the backbone chemical shifts by TALOS+ suggests that EF-P contains 1 α-helix and 13 β-strands (β1-β2-β3-β4-β5-β6-β7-α1-β8-β9-β10-β11-β12-β13). The solution of the structure of this protein by NMR and X-ray diffraction analysis, as well as the structure of the ribosome complex by cryo-electron microscopy, will allow further screening of highly selective inhibitors of the translation of the pathogenic bacterium S. aureus. Here we report the almost complete 1H, 13C, 15N backbone and side chain NMR assignment of a 20.5 kDa EF-P.

Background: Ribosome biogenesis is a complex process of ribosomal RNA and protein binding. Bacterial ribosome maturation and components involved in it are especially interesting, because they are widespread targets for antibiotics. A number of special protein factors facilitating the maturation of the 30S small ribosomal subunit are known. One of them is a ribosome-binding factor A (RbfA). This is a small (~14 kDa) protein with KH-domain organization distinguishing RNA binding proteins. Recent cryo-EM reconstruction of E.coli 30S-RbfA complex indicates that RbfA binds to 30S subunit on the central decoding region and promotes the switch from the immature state of h28 (neck) to mature state. RbfA interacts with 3`-end of 16S rRNA on mRNA exit channel and stabilizes the conformation of the region between h28, h44/h45 linker and 3`-end. Methods: Pure S.aureus RbfA was obtained by homologous expression in E.coli BL21 strain followed by Ni-NTA and gel filtration. The 30S subunits were obtained by dissociation of the S.aureus 70S ribosomes in a sucrose gradient (0-30%). We performed 30S subunit and RbfA complex reconstitution, sample and grid preparation. Data was collected on Talos Arctica, Falcon 2 detector (FEI Company/Thermo Fisher). Results: The 30S-RbfA complex density map with average resolution ~ 3.5 Å (FSC=0.143) was obtained. In comparison with the free subunit map (EMD 23052) we observed an extra density on the neck region near the decoding center region. Conclusion: Obtained data is correlated with recent structural results of the homologous E.coli RbfA. We consider that S.aureus RbfA binds to the 30S subunit at the same region. The next step of our structural research is building the model of S.aureus 30S-RbfA complex.

Mathematical simulation of m-phase synchronous motors (m≥3) is based on assumptions of a smooth air gap of the machine and there is no saturation of the magnetic circuit, which reduces the degree of correctness of their research vibration characteristics. Mathematical model for calculating the electromagnetic forces m-phase synchronous motor with permanent magnets is proposed, which takes into account the actual distribution of the magnetic field in the core of the machine, taking into account the saturation of the magnetic circuit, the discrete distribution of the multiphase stator windings and serration of the motor stator core.

Elongation factor P (EF-P) is a three domain protein that binds to the ribosome between P and E sites.The EF-P involved in a specialized translation of stalling amino acid motifs such as (PPP or APP). Proteins with stalling motifs are involved in various cell processes, including stress resistance and virulence of bacteria. EF-P stabilizes the P-tRNA and increases the entropy of stalled ribosome complex to compensate for rigid nature of proline residue, thus providing adequate protein synthesis rate. Detailed structural mechanisms of this effect are still poorly understood. It was shown that most of bacteria needs a special post-translational modification in a conservative region of the loop in the domain I of the EF-P located near the CCA-end of P-tRNA and the peptidyl transferase center. In present paper by EPR spectroscopy we investigated a spinlabeled EF-P from Staphylococcus aureus—a pathogenic bacteria which causes various human diseases. Addition of MTSL ((S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate)) label covalently bound to 32Cys in the highly conservative part of EF-P loop in the domain I of the EF-P allows us to analyze protein dynamic by EPR spectroscopy of the high mobility region which was not observed in NMR spectra due to fast proton exchange leading to the absence of the corresponding amide NMR resonances. We suppose that our result could be extended in future for the analysis of EF-P—ribosome complex formation process by advanced EPR techniques.

Temperature is a major environmental factor that influences all aquatic organisms. Our objective was to expose individuals of Lymnaea stagnalis from an Eastern Siberian population to wide temperature gradients to determine whether they express a thermal stress response and what mechanisms are involved in this process. Several parameters of the cellular mechanisms of thermal resistance were tested under the experimental conditions: the level of lipid peroxidation, the synthesis of heat shock protein HSP70, the activities of antioxidative enzymes and changes in the content of energy-associated metabolites. The results of this study show that the temperature at which the cellular and biochemical stress-response is activated in the L. stagnalis from Eastern Siberian lakes directly corresponds to environmental temperature within the range of 3 to 15°C. The exposure of the gastropods to temperatures outside this range resulted in the activation of HSP70 synthesis, changes in the activities of antioxida...