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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.

Background: Antibiotic resistance is a growing worldwide problem. One of the major resistant bacterial pathogens is Staphylococcus aureus, which became a burden of healthcare systems around the world. To overcome the issue, more drug discovery studies are needed. One of the main antibiotic targets is a ribosome – the central hub of protein synthesis. Structural data of the ribosome and its features are a crucial milestone for the effective development of new drugs, especially using structure-based drug design approaches. Apart from many small structural features, ribosome possesses rRNA modifications that play a role in the fine-tuning of protein synthesis. Detailed species-specific structural data of the S. aureus ribosome is also a useful model for understanding the resistance mechanisms. This information could help with the design of new antibiotics and the upgrading of old ones. The data on S. aureus ribosomal RNA modifications and corresponding modification enzymes are very limited. Our aim was to improve the current models of the S. aureus ribosome by determining its structure with functional ligands at a much higher resolution - thereby creating a foundation for structure-based drug design experiments and research of new drug targets. Methods: The S. aureus ribosome complex consists of three components: ribosome, fMet-tRNAfMet, mRNA and 70S ribosome. The complex from purified components was formed in vitro and applied to cryo-EM grids. Data was collected at Titan Krios with Gatan K2 detector (IGBMC, France). The data was processed and modeled in Relion 2.1, Chimera, Coot, and Phenix. Results: We determined the cryo-EM reconstruction at 3.2 Å resolution of the S. aureus ribosome with P-site tRNA, messenger RNA. Based on the experimental map and existing bioinformatic data, we at the first time identified and assigned 10 modifications of S. aureus rRNA. We analyzed the positions of rRNA modifications and their possible functions. Conclusion: In this study, we describe our structure of S. aureus ribosome with functional ligands. The present model is the highest resolution and most precise that is available at the moment. We propose a set of methyltransferases as targets for future drug discovery studies. The proposed methyltransferases and corresponding modifications may play an important role in protein synthesis and its regulation.

Background: In bacteria, diminishing of protein synthesis is often accompanied with the formation of ribosomal dimers (or disomes). This process might be particularly important for pathogenic bacteria whose successful survival is a key factor for successful infection. Ribosome hibernation is driven by the binding of small stress-induced protein(s) to 70S ribosomes forming stable 100S ribosomal dimers. In contrast with E. coli where ribosome dimerization occurs in the presence of two proteins: hibernation promoting factor (HPF, 95 aa residues) and ribosome modulation factor (RMF, 55 aa residues) in Staphylococcus aureus only one protein (long version of HPF) is responsible for the formation of hibernating ribosome dimers, which in turn play a role in survival of this pathogen under unfavorable conditions. Methods: By cryo-electron microscopy we obtained a high-resolution structure of the 100S ribosome dimer and by NMR spectroscopy and X-ray crystallography solved a NTD and CTD of SaHPF protein structures. Results: Our recent cryo-EM studies demonstrated that integrity of 100S disomes is maintained primarily by interactions between C-terminal domains (CTD) of SaHPF with each other. N-terminal domain (NTD) of SaHPF binds to the 30S subunit at the P-site and A-site and may inhibit protein synthesis as observed for shorter variants of HPF in other species. By NMR spectroscopy we have determined the solution structure of SaHPF-NTD and analyzed its binding with S. aureus 70S ribosome. Conclusion: Based on NMR and crystal structures of SaHFP-CTD we predicted the key amino acids residues stabilizing dimer interface and made several single amino acid substitutions which allows us to abolish the ribosome hibernation. Obtained information about crucial for dimerization residues in long HPF may be used as a potential target for combating S. aureus.