Abstract P-31: Assembly of the Complex of the 30S Ribosomal Subunit and the Ribosome Maturation Factor P from Staphylococcus aureus for Structural Studies by Cryo-Electron Microscopy

Background: Staphylococcus aureus (S. aureus) is one of the main human pathogens causing numerous nosocomial soft tissue infections and is among the best-known causes of bacterial infections. The bacterial 70S ribosome consists of two subunits, designated the 30S (small) and 50S (large) subunits. The small subunit (30S) consists of 16S ribosomal RNA (rRNA), from which the assembly of 30S begins, and 21 ribosomal proteins (r-proteins). The ribosome maturation factor P (RimP protein) binds to the free 30S subunit. Strains lacking RimP accumulate immature 16S rRNA, and fewer polysomes and an increased amount of unassociated 30S and 50S subunits compared to wild-type strains are observed in the ribosomal profile. Structural studies of the 30S subunit complex and the ribosome maturation factor RimP will make it possible in the future to develop an antibiotic that slows down or completely stops the translation of Staphylococcus aureus, which will complicate the synthesis and isolation of its pathogenic factors. Here we present the protocol of the in vitro reconstruction of S. aureus 30S ribosome subunit in a complex with RimP for further structural studies by cryo-electron microscopy. Methods: Recombinant RimP protein from S. aureus was expressed in E. coli and purified by Ni-NTA chromatography and size exclusion chromatography. Reconstitution of the 30S–RimP complex was performed by mixing RimP protein with 30S ribosome. Unbound RimP protein was removed by Amicon Ultra Concentration (Merk KGaA, Darmstadt, Germany) with a cut-off limit of 100 kDa. The presence of RimP protein in the resulting 30S-RimP complex was confirmed by SDS-PAGE, and the quality of the final sample was analyzed by the negative staining EM. Results: Finally, by in vitro reconstruction, the 30S-RimP complex from S. aureus was obtained for further structural studies by cryo-electron microscopy

Abstract P-26: Staphylococcus Aureus 30S Ribosomal Subunit in a Complex with the Era GTPase: Sample Preparation for Cryo-EM

Background: An essential in bacteria GTPase Era is a multifunctional protein that is involved in cell cycle regulation and appears to play a significant role in ribosome biogenesis. It is required for the maturation of the 30S ribosomal subunit. Era consists of two domains: the GTPase N-terminal domain, conserved in the GTPase family, and a C-terminal RNA-binding KH domain. Era specifically binds to the 16S rRNA and stimulates processing of the small ribosomal subunit to its mature form. Precise determination of nucleotide and amino acid sequences in the active site of binding will help in finding specific ways to prevent this interaction. In this way, it will be possible to disrupt the biogenesis of the ribosome and, thereby, stop or slow down protein synthesis in the bacterial cell. It is very important in the fight against pathogenic bacteria, such as Staphylococcus aureus (S. aureus). Methods: The His-tagged Era (His–Era) protein from S. aureus was expressed in E. coli BL21 strain and purified by Ni-NTA and SEC. The 30S ribosomal subunits were collected after dissociation of the S. aureus 70S ribosomes in sucrose gradient (0 – 30%). Complex 30S-Era was obtained by mixing in vitro 30S subunits and His–Era, incubated for 15 min at 37°C and followed by Ni-NTA purification to remove unbound 30S subunits. The presence of a stable 30S-Era complex has been confirmed by SDS-PAGE and agarose gel electrophoresis. The final sample quality was analyzed by negative staining EM. Results: For the first time in vitro 30S-Era complex from S. aureus was assembled and a sample was prepared for further structural studies by cryo-electron microscopy

Abstract P-21: The Investigation of S.aureus Ribosome-Binding Factor A Localization on the 30S Ribosomal Subunit by Cryo-Electron Microscopy

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

Yet Another Similarity between Mitochondrial and Bacterial Ribosomal Small Subunit Biogenesis Obtained by Structural Characterization of RbfA from S. aureus

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

Yet Another Similarity between Mitochondrial and Bacterial Ribosomal Small Subunit Biogenesis Obtained by Structural Characterization of RbfA from S. aureus

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

Dimerization of long hibernation promoting factor from Staphylococcus aureus: Structural analysis and biochemical characterization

International audienceStaphylococcus aureus hibernation promoting factor (SaHPF) is responsible for the formation of 100S ribosome dimers, which in turn help this pathogen to reduce energy spent under unfavorable conditions. Ribosome dimer formation strongly depends on the dimerization of the C-terminal domain of SaHPF (CTDSaHPF). In this study, we solved the crystal structure of CTDSaHPF at 1.6 Å resolution and obtained a precise arrangement of the dimer interface. Residues Phe160, Val162, Thr171, Ile173, Tyr175, Ile185 andThr187 in the dimer interface of SaHPF protein were mutated and the effects were analyzed for the formation of 100S disomes of ribosomes isolated from S. aureus. It was shown that substitution of any of single residues Phe160, Val162, Ile173, Tyr175 and Ile185 in the SaHPF homodimer interface abolished the ribosome dimerization in vitro