An RNA structural switch controlling bacterial toxin translation.

Type I toxin-antitoxin systems (T1TAs) rely on tight posttranscriptional control to prevent inadvertent toxin synthesis, yet the molecular mechanisms underlying this control are highly diverse. Here, we uncover an RNA-based mechanism that controls translation initiation in the enterobacterial timPR system. Unlike most T1TAs, which typically rely on ribonucleolytic messenger RNA (mRNA) processing to relieve ribosome binding site sequestration, the primary timP toxin mRNA is activated through a purely structural RNA switch. Using a FASTBAC-Seq loss-of-function screen with biochemical and phenotypic assays, we here identify key RNA interactions that govern this switch. Translation initiation at timP requires formation of (i) a pseudoknot in the 5' untranslated region, and (ii) a long-range interaction that destabilizes the ribosome-binding-site-sequestering stem-loop, rendering the Shine-Dalgarno sequence accessible for pre-initiation complex formation. Conversely, an alternative interaction locks the mRNA in an inactive state. Our findings reveal a structural RNA switch that controls toxin expression without the need for enzymatic processing and demonstrate an alternative mechanism for translation initiation in bacteria.