Antisense RNA regulation of the par post-segregational killing system: structural analysis and mechanism of binding of the antisense RNA, RNAII and its target, RNAI

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 RNAI and RNAII, which are the toxin and antito xin of the par PSK system respectively. RNAI encodes an open reading frame for a 33 amino acid toxin called Fst. Expression of fst is regulated posttr anscriptionally by RNAII. RNAII interacts with RNAI by a unique antisense R NA mechanism involving binding at the 5' and 3' ends of both RNAs. Par RNA interaction requires a complementary transcriptional terminator stem-loop a nd a set of direct repeat sequences, DRa and DRb, located at the 51 end of both RNAs. The secondary structures of RNAI, RNAII and the RNAI-RNAII compl ex were analysed by partial digestion with Pb(II) and ribonucleases. Probin g data for RNAII and RNAII are consistent with previously reported computer generated models, and also confirm that complementary direct repeat and te rminator sequences are involved in the formation of the RNAI-RNAII complex. Mutant par RNAs were used to show that the binding reaction occurs in at l east two steps. The first step is the formation of an initial kissing inter action between the transcriptional terminator stem-loops of both RNAs. The subsequent step(s) involves an initial pairing of the complementary direct repeat sequences followed by complete hybridization of the 51 nucleotides t o stabilize the RNAI-RNAII complex.