Mechanisms of Action of Ribosome-Targeting Antibiotics

The ribosome is a ribonucleoprotein complex responsible for synthesizing proteins from mRNA in a process called translation. Differences between prokaryotic and eukaryotic ribosomes make the ribosome a significant target for antibiotics. Within this dissertation, the mechanisms of action of three ribosome-targeting antibiotics are studied using next generation sequencing, molecular biology, and biochemistry. We first reveal the surprising in-cell mechanism of action of the orthosomycin antibiotic evernimicin. Evernimicin’s action is context-specific, meaning it most efficiently inhibits translation when certain amino acids are in the ribosome. Using this knowledge, we identify a short open reading frame that may be involved in the regulation of the orthosomycin resistance gene, EmtA. Next, we describe the genome-wide effects of apidaecin, a proline-rich antimicrobial peptide, and the first described specific inhibitor of translation termination. We show that in cells, apidaecin sequesters release factors and traps ribosomes at stop codons. There are also significant secondary effects, including stop codon readthrough and overwhelming the ribosome rescue systems. Finally, we demonstrate that drosocin, another proline-rich antimicrobial peptide, is the second described specific inhibitor of translation termination. A plasmid library containing the coding sequence of 362 arabinose-inducible drosocin gene variants was used to analyze sequence activity relationships of drosocin.