Mechanism of Post-segregational Killing: Secondary Structure Analysis of the Entire Hok mRNA from Plasmid R1 Suggests a Fold-back Structure that Prevents Translation and Antisense RNA Binding

Abstract

Thehok/soksystem of plasmid R1 mediates plasmid stabilization by killing of plasmid-free cells. The Hok mRNA is very stable and can be translated into Hok killer protein. Translation of the Hok mRNA is inhibited by the small unstable Sok antisense RNA. Translation ofhokis coupled to an overlapping reading frame termedmok. Translation ofmokis tightly regulated by Sok RNA, and Sok RNA thus regulateshoktranslation indirectly throughmok. The rapid decay of Sok RNA explains the onset of Hok synthesis in newborn plasmid-free segregants. However, a second control level is superimposed on this simple induction scheme, since the full-length Hok mRNA was found to be translationally inactive whereas a 3′-end truncated version of it was active. We have therefore previously suggested, that the 3′-terminal region of the full-length Hok mRNA encodes an element which prevents its translation. This element was termedfbi(fold-back inhibition). Here we describe thein vitrosecondary structure of the entire Hok mRNA. Our results suggest a closed structure in which the 3′-end of the full-length Hok mRNA folds back onto the translational initiation region ofmok. This structure explains why full-length Hok mRNA is translationally silent. The proposed structure was further supported by results obtained using mutations in the 3′-endfbielement. These “structure closing” mutations affected the structure much further upstream in themoktranslational initiation region and concomitantly prevented antisense RNA binding to the same region of the mRNA. These results lend further support to the induction model that explains onset of Hok mRNA translation in plasmid-free segregants. The most important regulatory element in this model is the FBI structure formed between the 3′-end and themoktranslational initiation region. This structure renders Hok mRNA translationally inactive and prevents antisense RNA binding, thus allowing the accumulation of a pool of mRNA which, by slow 3′-end processing, is activated in plasmid-free segregants, eventually leading to the elimination of these cells.