Project Summary/Abstract Elongating ribosomes frequently encounter obstacles that cause ribosomes to stall. Stalled ribosomes are then targeted by rescue factors to recycle the ribosomal subunits and target the faulty mRNA and nascent peptide for decay. This process is critical for cellular fitness in bacteria: its loss often results in decreased pathogenicity, viability, or antibiotic resistance. In E. coli, ribosome stalling leads to collisions, which recruit the endonuclease SmrB to cleave the mRNA between collided ribosomes. These ribosomes are then targeted by the rescue factor tmRNA. Several lines of evidence suggest E. coli employ a second pathway to split antibiotic- inhibited ribosomes into subunits; however, a ribosome-splitting factor has yet to be identified. The DExH-box RNA helicase HrpA decreases E. coli sensitivity to ribosome-targeting antibiotics, suggesting this protein has a ribosome-associated function in antibiotic resistance. In fact, HrpA has been shown to rescue ribosomes stalled on reporter mRNA and resolve global antibiotic-induced ribosome collisions, indicating that it plays a role in clearing stacked ribosomes during translational stress. The proposed experiments will explore whether HrpA is a novel ribosome rescue factor that splits stalled ribosomes. Because ribosome collisions are critical for ribosome rescue in yeast, mammalian cells, and E. coli, Aim 1 will determine if ribosome collisions also recruit HrpA. Protein readout from a reporter-based assay in wild-type and ΔhrpA cells will be used to test if HrpA rescues collided ribosomes from mRNA. To determine if HrpA preferentially associates with collided ribosomes in vivo, sucrose density gradients will be used to determine if HrpA sediments with ribosome subunits, single ribosomes, or collided polysomes from cell lysates that are treated with ribosome-stalling antibiotics. Finally, the impact of HrpA on ribosome position and ribosome collisions transcriptome-wide will be explored using ribosome profiling in antibiotic-treated wild-type and ΔhrpA cells. Two S. cerevisiae RNA helicases in the same DExH-box family as HrpA are involved in ribosome splitting; therefore, Aim 2 will determine if HrpA similarly splits stalled ribosomes in E. coli. Because the protein Hsp15 preferentially associates with 50S ribosomal subunits from prematurely split ribosomes, HrpA splitting activity will be assayed in vivo by examining Hsp15 sedimentation in sucrose density gradients in antibiotic-treated wild-type and ΔhrpA cells. Finally, HrpA will be tested for its ability to split stalled ribosomes into subunits using an in vitro biochemical assay with purified components analyzed in sucrose density gradients. This assay will be used to determine ATPase activity and precise substrate recognition of HrpA. Characterization of HrpA will provide valuable insight into how E. coli mitigate the effects of ribosome-targeting antibiotics, marking it as a promising potential target of anti...