Search

Summary Through targeted inactivation of the ssrA and smpB genes, we establish that the trans-translation process is necessary for normal growth, adaptation to cellular stress and virulence by the bacterial pathogen Francisella tularensis. The mutant bacteria grow slower, have reduced resistance to heat and cold shocks, and are more sensitive to oxidative stress and sublethal concentrations of antibiotics. Modifications of the tmRNA tag and use of higher-resolution mass spectrometry approaches enabled the identification of a large number of native tmRNA substrates. Of particular significance to understanding the mechanism of trans-translation, we report the discovery of an extended tmRNA tag and extensive ladder-like pattern of endogenous protein-tagging events in F. tularensis that are likely to be a universal feature of tmRNA activity in eubacteria. Furthermore, the structural integrity and the proteolytic function of the tmRNA tag are both crucial for normal growth and virulence of F. tularensis. Significantly, trans-translation mutants of F. tularensis are impaired in replication within macrophages and are avirulent in mouse models of tularemia. By exploiting these attenuated phenotypes, we find that the mutant strains provide effective immune protection in mice against lethal intradermal, intraperitoneal and intranasal challenges with the fully virulent parental strain.

The translation machinery deciphers genetic information encoded within mRNAs to synthesize proteins needed for various cellular functions. Defective mRNAs that lack in-frame stop codons trigger non-productive stalling of ribosomes. We investigated how cells deal with such defective mRNAs, and present evidence to demonstrate that RNase R, a processive 3'-to-5' exoribonuclease, is recruited to stalled ribosomes for the specific task of degrading defective mRNAs. The recruitment process is selective for non-stop mRNAs and is dependent on the activities of SmpB protein and tmRNA. Most intriguingly, our analysis reveals that a unique structural feature of RNase R, the C-terminal lysine-rich (K-rich) domain, is required both for productive ribosome engagement and targeted non-stop mRNA decay activities of the enzyme. These findings provide new insights into how a general RNase is recruited to the translation machinery and highlight a novel role for the ribosome as a platform for initiating non-stop mRNA decay.

The SmpB-tmRNA-mediated trans-translation system has two well-established activities: rescuing ribosomes stalled on aberrant mRNAs and marking the associated protein fragments for proteolysis. Although the causative non-stop mRNAs are known to be degraded, little is known about the enabling mechanism or the RNases involved in their disposal. We report that Escherichia coli has an enabling mechanism that requires RNase R activity and is dependent on the presence of SmpB protein and tmRNA, suggesting a requirement for active transtranslation in facilitating RNase R engagement and promoting non-stop mRNA decay. Interestingly, this selective transcript degradation by RNase R targets aberrant (non-stop and multiple-rare-codon containing) mRNAs and does not affect the decay of related messages containing in-frame stop codons. Most surprisingly, RNase II and PNPase do not play a significant role in tmRNA-facilitated disposal of aberrant mRNAs. These findings demonstrate that RNase R is a crucial component of the trans-translation-mediated non-stop mRNA decay process, thus providing a requisite activity well suited to complement the ribosome rescue and protein tagging functions of this unique quality control system.