Determining the off-target activity of antibiotics and novel translation initiation sites in mitochondria

Mitochondria translate the 13 mitochondrially encoded proteins using a dedicated translation system that closely resembles the one found in prokaryotes. Consequently, many bacterial protein synthesis inhibitors, used as antibiotics, exhibit mitochondrial toxicity as off-target effects. However, it remains unclear whether these antibiotics act through the same mechanisms in mitochondria as in bacteria. To address this, we characterized the effects of a panel of bacterial translation and elongation inhibitors on mitochondrial translation through mitoribosome profiling. We found that the mechanism of action for every antibiotic, except telithromycin, is the same in both bacteria and mitochondria. Additionally, mitoribosome profiling data showed that MT-ND1 and MT-ND5 have incorrectly annotated translation initiation sites and suggested several novel mitochondrial translation events. Careful study of the mechanisms of mitochondrial translation inhibition can guide development of antibiotics with increased target specificity and reduced mitochondrial toxicity.