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

Abstract

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.