Programmed cell death by hok/sok of plasmid R1: Processing at the hok mRNA 3′-end triggers structural rearrangements that allow translation and antisense RNA binding

Abstract

The hok/sok locus of plasmid R1 mediates plasmid stabilization by killing of plasmid-free cells. The locus specifies two RNAs, hok mRNA and Sok antisense RNA. The post-segregational killing mediated by hok/sok is governed by a complicated control mechanism that involves both post-transcriptional inhibition of translation by Sok-RNA and activation of hok translation by mRNA 3′ processing. Sok-RNA inhibits translation of a reading frame ( mok) that overlaps with hok, and translation of hok is coupled to translation of mok. In the inactive full-length hok mRNA, the translational activator element at the mRNA 5′-end ( tac) is sequestered by the fold-back-inhibitory element located at the mRNA 3′-end ( fbi). The 5′ to 3′ pairing locks the RNA in an inert configuration in which the SD mok and Sok-RNA target regions are sequestered. Here we show that the 3′ processing leads to major structural rearrangements in the mRNA 5′-end. The structure of the refolded RNA explains activation of translation and antisense RNA binding. The refolded RNA contains an antisense RNA target stem-loop that presents the target nucleotides in a single-stranded conformation. The stem of the target hairpin contains SD mok and AUG mok in a paired configuration. Using toeprinting analysis, we show that this pairing keeps SD mok in an accessible configuration. Furthermore, a mutational analysis shows that an internal loop in the target stem is prerequisite for efficient translation and antisense RNA binding.