A New Mechanism for Ribosome Rescue Can Recruit RF1 or RF2 to Nonstop Ribosomes

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 “nonstop” ribosomes are rescued using trans-translation. Genes required for trans-translation are essential in some species, 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 (wt) cells but not in cells that lack trans-translation activity. Overexpression 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 nonstop ribosomes in vivo. Ribosome rescue assays in vitro show that ArfT promotes hydrolysis of peptidyl-tRNA on nonstop 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.