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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.

Ribosome binding factor A (RbfA) is a 14.9 kDa adaptive protein of cold shock, which is important for bacterial growth at low temperatures. RbfA can bind to the free 30S ribosomal subunit and interacts with the 5'-terminal helix (helix I) of 16S rRNA. RbfA is important for the efficient processing of 16S rRNA and for the maturation (assembly) of 30S ribosomal subunits. Here we report backbone and side chains (1)H, (13)C and (15)N chemical shift assignments of RbfA from Staphylococcus aureus. Analysis of the backbone chemical shifts by TALOS+ suggests that RbfA contains four alpha-helixes and three beta-strands with alpha1-beta1-beta2-alpha2-alpha3-beta3-alpha4 topology. Secondary structure of RbfA have KH-domain fold topology with betaalphaalphabeta subunit which is characterized by a helix-kink-helix motif in which the GxxG sequence is replaced by a conserved AxG sequence, where an Ala residue at position 70 forming an interhelical kink. The solution of the structure of this protein factor and its complex with the ribosome by NMR spectroscopy, X-ray diffraction analysis and cryo-electron microscopy will allow further development of highly selective substances for slowing or completely stopping the translation of the pathogenic bacterium S. aureus, which will interfere with the synthesis and isolation of its pathogenicity factors.

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: 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.

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.