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

The 13 mtDNA-encoded proteins are synthesized using a dedicated translation system that is more similar to bacterial systems than the cytoplasmic system. Consequently, many bacterial protein synthesis inhibitors, used as antibiotics, exhibit mitochondrial toxicity as off-target effects. However, whether these antibiotics act through the same mechanisms in mitochondria as in bacteria remains unclear. To address this, we characterized the impact of a panel of bacterial translation initiation 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 the presence of several translation initiation sites on ncRNAs that produce mitoribosome footprints, as indicated by the detection of mitoribosome footprints at these locations. This work demonstrates how antibiotics can inhibit mitochondrial translation by mechanisms identically or very similar to those found in bacteria and the utility of mitoribosome profiling for annotating mitochondrial genes.