A bacterial regulatory uORF senses multiple classes of ribosome-targeting antibiotics

Expression of many bacterial genes is regulated bycis- andtrans-acting elements in their 5’ upstream regions (URs).Cis-acting regulatory elements in URs include upstream ORFs (uORF), short ORFs that sense translation stress that manifests as ribosomes stalling at specific codons within the uORF. Here, we show that the transcript encoding theEscherichia coliTopAI-YjhQ toxin-antitoxin system is regulated by a uORF that we name “toiL”. We propose that in the absence of translation stress, a secondary structure in the UR represses translation of thetopAItranscript by occluding the ribosome-binding site. Translation repression oftopAIleads to premature Rho-dependent transcription termination within thetopAIORF. At least five different classes of ribosome-targeting antibiotics relieve repression oftopAI. Our data suggest that these antibiotics function by stalling ribosomes at different positions withintoiL, thereby altering the RNA secondary structure around thetopAIribosome-binding site. Thus,toiLis a multipurpose uORF that can respond to a wide variety of translation stresses.