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.

Abstract

The par stability determinant of the Enterococcus faecalis plasmid pAD1 is the first antisense RNA regulated post-segregational killing system (PSK) identified in a Gram-positive organism. Par encodes two small, convergently transcribed RNAs, designated RNAI and RNAII, which are the toxin and antitoxin 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 post-transcriptionally by RNAII. RNAII interacts with RNAI by a unique antisense RNA mechanism involving binding at the 5' and 3' ends of both RNAs. Par RNA interaction requires a complementary transcriptional terminator stem-loop and a set of direct repeat sequences, DRa and DRb, located at the 5' end of both RNAs. The secondary structures of RNAI, RNAII and the RNAI-RNAII complex were analysed by partial digestion with Pb(II) and ribonucleases. Probing data for RNAI and RNAII are consistent with previously reported computer generated models, and also confirm that complementary direct repeat and terminator 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 least two steps. The first step is the formation of an initial kissing interaction 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 5' nucleotides to stabilize the RNAI-RNAII complex.