In bacteria, trans-translation is the main quality control mechanism for rescuing ribosomes arrested during translation. This key process is universally conserved and plays a critical role in the viability and virulence of many pathogens. We developed a reliable in vivo double-fluorescence reporter system for the simultaneous quantification of both trans-translation and the associated proteolysis activities in bacteria. The assay was validated using mutant bacteria lacking tmRNA, SmpB, and the ClpP protease. Both antisense tmRNA-binding RNA and a peptide mimicking the SmpB C-terminal tail proved to be potent inhibitors of trans-translation in vivo. The double-fluorescent reporter was also tested with KKL-35, an oxadiazole derivative that is supposed to be a promising trans-translation inhibitor, and it surprisingly turns out that trans-translation is not the only target of KKL-35 in vivo.
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Article Synopsis
- Ribosomes in bacteria often deal with incomplete or damaged mRNAs, and when they encounter non-stop mRNAs (lacking a stop codon), specialized rescue pathways are necessary to free them.
- The most common method for this rescue is known as -translation, found in over 95% of bacterial genomes, while in Proteobacteria, proteins ArfA and ArfB play crucial roles when -translation is absent.
- The study introduces RqcH, a ribosome quality control factor that tags stalled peptides to help clear them from the ribosome, suggesting that RqcH aids non-stop ribosome rescue and highlighting diverse rescue pathways in over 14,000 bacterial genomes.
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