MECHANISM OF POST-SEGREGATIONAL KILLING - SECONDARY STRUCTURE-ANALYSIS OF THE ENTIRE HOK MESSENGER-RNA FROM PLASMID R1 SUGGESTS A FOLDBACK STRUCTURE THAT PREVENTS TRANSLATION AND ANTISENSE RNA-BINDING

The hok/sok system of plasmid R1 mediates plasmid stabilization by kil ling of plasmid-free cells. The Hok mRNA is very stable and can be tra nslated into Hok killer protein. Translation of the Hok mRNA is inhibi ted by the small unstable Sok antisense RNA. Translation of hok is cou pled to an overlapping reading frame termed mok. Translation of mok is tightly regulated by Sok RNA, and Sok RNA thus regulates hok translat ion indirectly through mok. The rapid decay of Sok RNA explains the on set of Hok synthesis in newborn plasmid-free segregants. However, a se cond control level is superimposed on this simple induction scheme, si nce 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 eleme nt was termed fbi (fold-back inhibition). Here we describe the in vitr o secondary structure of the entire Hok mRNA. Our results suggest a cl osed structure in which the 3'-end of the full-length Hok mRNA folds b ack onto the translational initiation region of mok. This structure ex plains why full-length Hok mRNA is translationally silent. The propose d structure was further supported by results obtained using mutations in the 3'-end fbi element. These ''structure closing'' mutations affec ted the structure much further upstream in the mok translational initi ation region and concomitantly prevented antisense RNA binding to the same region of the mRNA. These results lend further support to the ind uction model that explains onset of Hok mRNA translation in plasmid-fr ee segregants. The most important regulatory element in this model is the FBI structure formed between the 3'-end and the mok translational initiation region. This structure renders Hok mRNA translationally ina ctive and prevents antisense RNA binding, thus allowing the accumulati on of a pool of mRNA which, by slow 3'-end processing, is activated in plasmid-free segregants, eventually leading to the elimination of the se cells.