Role of the DEAD-box helicases CshA and CshB in bacterial translational quality control

• Applicant: Alan Rodriguez Carvajal, Ph.D.
• Subject Area: Structural Biology ; Biochemistry ; Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 503872807

Properly functional translation is a fundamental necessity for all living cells.Many translational stresses cause ribosomes to stall. Trailingribosomes collide with the stalled leading ribosome to form a unique disomestructure. Collided disomes act as signals to activate ribosome rescuepathways and the downstream Ribosome Quality Control (RQC) pathway, whichfunction to degrade the incomplete nascent chain and recycle ribosomal sub-units. Our lab discovered the bacterial RQC pathway, which is homologous toeukaryotic RQC pathways; more recently our lab has characterized the novelcomponent MutS2 that senses collided disomes in bacteria. During the analysisof MutS2, the RNA helicases CshA and CshB were identified asputative ribosome rescue factors. In this project we aim to characterize the roleof CshA and CshB in bacterial ribosome rescue and RQC. Our first objective is toestablish whether the helicases function in ribosome splitting or mRNA decayin response to ribosome collisions. We will first generate knock-out strains of thehelicases to establish a genetic connection with ribosome rescue and RQC. AsRNA helicases CshA and CshB may be involved in ribosome splitting or they maybe components of mRNA degradation downstream of ribosome rescue. Todetermine what their function is we will characterize the association of thehelicases to the ribosome by density centrifugation experiments and test theirability to split collided disomes in vitro. We will also test the stability of stall-inducing reporter mRNA in the absence of CshA and CshB. The second objectiveof this work is to determine the structures of CshA and CshB bound to ribosomalparticles by cryo-electron microscopy. To this end, we will purify helicase-boundribsomes by immuniprecipitation of tagged CshA and CshB for samplepreparation. We will take advantage of the ribosome's well-characterizedstructure to identify additional densities corresponding to the helicases. Atpresent we have confirmed the association of both helicases with the ribosomeand have obtained our first 3D reconstructions from CshAimmunoprecipitations. Due to their evolutionary conservation, characterizationof bacterial ribosome rescue and RQC could provide further insight into thehomologous eukaryotic pathways, which have been implicated inneurodegenerative disease. Furthermore, components of bacterial ribosomerescue and RQC may be good candidates for development of novel therapeuticcompounds for use in combinatorial antibiotic treatments.

• DFG Programme: WBP Position