MECHANISM OF POST-SEGREGATIONAL KILLING BY HOK-HOMOLOG PND OF PLASMIDR483 - 2 TRANSLATIONAL CONTROL ELEMENTS IN THE PND MESSENGER-RNA

The pnd system of plasmid R483 mediates plasmid stabilization by killi ng of plasmid-free cells. The pnd mRNA is very stable and can be trans lated into PndA protein, a cell toxin which kills the cells from withi n by damaging the cell membrane. Translation of the pnd mRNA is inhibi ted by the PndB antisense, a small labile RNA of 63 nt. The rapid deca y of the PndB antidote leads to onset of PndA synthesis in plasmid-fre e segregants or after addition of rifampicin. Surprisingly however, th e full-length pnd mRNA was found to be translationally inactive wherea s a 3'-end truncated version of it was found to be active. We have the refore suggested previously, that the 3'-end of the full-length pnd mR NA encodes a fold-back inhibitory sequence (fbi), which prevents its t ranslation. Here we present an analysis of the metabolism of the pnd m RNAs. A mutational analysis shows that single point mutations in the f bi motif results in more rapid truncation. The fbi mutations could not be complemented by second-site mutations in either of the pndA or pnd C Shine-Dalgarno (SD) elements. Surprisingly, mutations in the pndC SD element also lead to a more rapid truncation. The effect of these lat ter mutations was, however, complemented by mutations in a proposed an ti-SD element upstream of the pndC SD. Mutations in the anti-SD elemen t were lethal. These results show, that the pnd mRNA contains two nega tive control elements, one located in its very 3'-end (fbi), and one l ocated just upstream of the pndC SD region (the anti-SD element). Thes e observations add to the complexity of the induction scheme previousl y proposed to explain activation of pndA expression in plasmid-free ce lls: In addition to its negative effect of translation, the fbi struct ure also maintains a reduced processing rate in the S'-end of the mRNA . This permits the accumulation of a reservoir of pnd mRNA, which can be activated by 3'-end processing in plasmid-free cells. The anti-SD m ay prevent translation of the pnd mRNA during transcription, thus prev enting detrimental synthesis of toxin.