Role of the DEAD-box helicases CshA and CshB in bacterial translational quality control
Biochemistry
Metabolism, Biochemistry and Genetics of Microorganisms
Final Report Abstract
Ribosome collisions are translation stress signals, In B. subtilis these are recognized by MutS2 which splits the leading ribosome. However, current structural data indicates that MutS2 can only act on collisions where the stalled leader is in an unrotated conformation, but not on collisions with rotated leaders or stalled monosomes. Our lab previously identified two RNA helicases, CshA and CshB, enriched in immunoprecipitations of MutS2. RNA helicases split ribosomes in collisions during eukaryotic translation stress response. Moreover, CshA has been functionally connected to the RNA degradosome of B. subtilis, suggesting that the helicases could be involved in mRNA degradation or ribosome splitting following ribosome collisions. In this work we found that deletion of the cshA or cshB genes sensitizes cells to antibiotics that stall ribosomes in a rotated conformation. Critically, deletion of cshA sensitizes cells to low doses of spectinomycin while deletion of cshB only sensitizes cells to high doses of spectinomycin that are too high to induce collisions. We further show that the sedimentation of CshA and CshB with ribosomes and collisions is affected by low and high doses of spectinomycin respectively and that this involves unidentified post-translational modifications of both helicases. Lastly, we found a novel ribosome collision state with the disome adopting a Top-to-Bottom orientation. While we were not able to biochemically characterize the precise function of the helicases, we establish a functional role for both CshA and CshB in bacterial translational quality control. We propose a model where CshA is involved in rescuing ribosome collisions with leaders stalled in a rotated state while CshB is involved in rescuing stalled monosomes.
