The antisense RNA of the par locus of pAD1 regulates the expression of a 33-amino-acid toxic peptide by an unusual mechanism

The par stability determinant of the Enterococcus faecalis plasmid pAD1 is the first antisense RNA-regulated post-segregational killing system (PSK) i dentified in a Gram-positive organism. Par encodes two small, convergently transcribed RNAs, designated RNA I and RNA II, which are the toxin and anti dote of the par PSK system respectively. RNA 1 encodes an open reading fram e of 33 codons designated ist The results presented here demonstrate that t he peptide encoded by fst is the par toxin, The fst sequence was shown to b e sufficient for cell killing, and removal of the final codon inactivated t he toxin. In vitro translation reactions of purified RNA I transcript produ ced a product of the expected size for the ist-encoded peptide. This produc t was not produced when purified RNA II transcript was added to the transla tion reaction. Toeprint analysis demonstrated that purified RNA II was able to inhibit ribosome binding to RNA I. These data suggest that fst expressi on is regulated by RNA II via an antisense RNA mechanism. In vitro translat ion studies and toeprint analyses also indicated that fst expression is int ernally regulated by a stem-loop structure at the 5' end of RNA I. Removal of this structure resulted in better ribosome binding to RNA I and a 300-fo ld increase in production of the fst-encoded peptide. Finally, RNA II was s hown to be less stable than RNA I in vivo, providing a basis for the select ive expression of ist in plasmid-free cells.