A new mechanism for ribosome rescue can recruit RF1 or RF2 to non-stop ribosomes

Abstract

Bacterial ribosomes frequently translate to the 3 end of an mRNA without terminating at an in-frame stop codon. In all bacteria studied to date, these non-stop ribosomes are rescued using trans-translation. In some species, genes required for trans-translation are essential, but other species can survive without trans-translation because they express an alternative ribosome rescue factor, ArfA or ArfB. Francisella tularensis cells lacking trans-translation are viable, but F. tularensis does not encode ArfA or ArfB. Transposon mutagenesis followed by deep sequencing (Tn-seq) identified a new alternative ribosome rescue factor, now named ArfT. arfT can be deleted in wild-type cells but not in cells that lack trans-translation activity. Over-expression of ArfT suppresses the slow growth phenotype in cells lacking trans-translation and counteracts growth arrest caused by trans-translation inhibitors, indicating that ArfT rescues non-stop ribosomes in vivo. Ribosome rescue assays in vitro show that ArfT promotes hydrolysis of peptidyl-tRNA on non-stop ribosomes in conjunction with F. tularensis release factors. Unlike ArfA, which requires RF2 for activity, ArfT can function with either RF1 or RF2. Overall, these results indicate that ArfT is a new alternative ribosome rescue factor with a distinct mechanism from ArfA and ArfB.\n\nImportanceFrancisella tularensis is a highly infectious intracellular pathogen that kills more than half of infected humans if left untreated. F. tularensis has also been classified as a potential bioterrorism agent with the greatest risk for deliberate misuse. Recently, compounds that inhibit ribosome rescue have been shown to have antibiotic activity against F. tularensis and other important pathogens. Like all bacteria that have been studied, F. tularensis uses trans-translation as the main pathway to rescue stalled ribosomes. However, unlike most bacteria, F. tularensis can survive without any of the known factors for ribosome rescue. Our work identifies a F. tularensis protein, ArfT, that rescues stalled ribosomes in the absence of trans-translation using a new mechanism. These results indicate that ribosome rescue activity is essential in F. tularensis and suggest that ribosome rescue activity might be essential in all bacteria.