Western CEDAR

Intermolecular Interactions that Lead to the Activation and Inhibition of Ribosome-Dependent GTPases

Miller Hall

The majority of antibiotics prescribed today target either bacterial ribosomes or translation factors, which function to block the vital process of protein synthesis. Bacterial evolution has led to antibiotic resistance and has increased the need to better understand ribosome-translation factor interactions. Translation of a messenger RNA template into proteins by the ribosome is facilitated by several translation factors, many of which are GTPases that utilize the hydrolysis of guanosine 5’-triphosphate to exert their function. Necessary for GTPase recruitment and activation is ribosomal protein L7/L12, which binds these translation factors with a highly conserved sequence in its C-terminal domain. This work aims to identify key residues in the conserved binding region of L7/L12 that are critical for GTPase binding and activation. Based on careful structural study of the L7/L12-GTPase binding interface, single amino acid point mutations (L7/L12-K66A, -K66D, -K82A, -K82D, -K85A, -K85D, -T77W, and -T77A) were generated by site-directed mutagenesis to asses functionality of conserved residues. By an established L7/L12 depletion protocol, wild type L7/L12 will be removed, followed by reconstitution of L7/L12 mutant proteins to ribosomal complexes. A sucrose cushion ultracentrifugation protocol will be used to assess ribosome-dependent GTPase binding, while GTPase activity will be quantified with a phosphate detection assay.