Structural analysis of the Anti-Q-Qs interaction: RNA-mediated regulation of E. faecalis plasmid pCF10 conjugation.

Conjugation of the E. faecalis plasmid pCF10 is triggered in response to peptide sex pheromone cCF10 produced by potential recipients. Regulation of this response is complex and multi-layered and includes a small regulatory RNA, Anti-Q that participates in a termination/antitermination decision controlling transcription of the conjugation structural genes. In this study, the secondary structure of the Anti-Q transcript and its sites of interaction with its target, Qs, were determined. The primary site of interaction occurred at a centrally-located loop whose sequence showed high variability in analogous molecules on other pheromone-responsive plasmids. This loop, designated the specificity loop, was demonstrated to be important but not sufficient for distinguishing between Qs molecules from pCF10 and another pheromone-responsive plasmid pAD1. A loop 5' from the specificity loop which carries a U-turn motif played no demonstrable role in Anti-Q-Qs interaction or regulation of the termination/antitermination decision. These results provide direct evidence for a critical role of Anti-Q-Qs interactions in posttranscriptional regulation of pCF10 transfer functions.

Direct evidence for control of the pheromone-inducible prgQ operon of Enterococcus faecalis plasmid pCF10 by a countertranscript-driven attenuation mechanism.

The mating response of Enterococcus faecalis cells carrying the conjugative plasmid pCF10 is controlled by multiple regulatory circuits. Initiation of transcription of the prgQ conjugation operon is controlled by the peptide receptor protein PrgX; binding of the pheromone peptide cCF10 to PrgX abolishes PrgX repression, while binding of the inhibitor peptide iCF10 enhances repression. The results of molecular analysis of prgQ transcripts and genetic studies suggested that the elongation of prgQ transcripts past a putative terminator (IRS1) may be controlled by the interaction of nascent prgQ mRNAs with a small antisense RNA (Anti-Q) encoded within prgQ. Direct evidence for interaction of these RNAs, as well as the resulting effects on readthrough of prgQ transcription, has been limited. Here we report the results of experiments that (i) determine the inherent termination properties of prgQ transcripts in the absence of Anti-Q; (ii) determine the direct effects of the interaction of Anti-Q with nascent prgQ transcripts in the absence of complicating effects of the PrgX protein; and (iii) begin to dissect the structural components involved in these interactions. The results confirm the existence of alternative terminating and antiterminating forms of nascent prgQ transcripts in vivo and demonstrate that the interaction of Anti-Q with these transcripts leads to termination via inhibition of antiterminator formation. In vitro transcription assays support the major results of the in vivo studies. The data support a model for Anti-Q function suggested from recent studies of these RNAs and their interactions in vitro (S. Shokeen, C. M. Johnson, T. J. Greenfield, D. A. Manias, G. M. Dunny, and K. E. Weaver, submitted for publication).

An intramolecular upstream helix ensures the stability of a toxin-encoding RNA in Enterococcus faecalis.

The par stability determinant is required for the stable inheritance of the plasmid pAD1 in its native host, Enterococcus faecalis. It is the only antisense RNA-regulated addiction module identified to date in gram-positive bacteria. It encodes two small, convergently transcribed RNAs, RNA I and RNA II. RNA I encodes the Fst toxin and RNA II acts as the antitoxin by interacting with RNA I posttranscriptionally. As the toxin-encoding component of the system, it is important that RNA I is more stable than RNA II. This study reveals that a helix sequestering the 5' end of RNA I plays a crucial role in maintaining the stability of the RNA I. An adjacent structure previously determined to regulate Fst translation was not required to enhance stability. Results indicated that endoribonuclease J2 contributes significantly to the degradation of a mutant disrupting the upstream helix (UH) of RNA I in Bacillus subtilis. Finally, it was shown that interaction with RNA II stabilized the UH mutant of RNA I.

Translational regulation by an intramolecular stem-loop is required for intermolecular RNA regulation of the par addiction module.

The par stability determinant of Enterococcus faecalis plasmid pAD1 is the only antisense RNA-regulated addiction module identified to date in gram-positive bacteria. par encodes two small, convergently transcribed RNAs, designated RNA I and RNA II, that function as the toxin (Fst)-encoding and antitoxin components, respectively. Previous work showed that structures at the 5' end of RNA I are important in regulating its translation. The work presented here reveals that a stem-loop sequestering the Fst ribosome binding site is required for translational repression but a helix sequestering the 5' end of RNA I is not. Furthermore, disruption of the stem-loop prevented RNA II-mediated repression of Fst translation in vivo. Finally, although Fst-encoding wild-type RNA I is not toxic in Escherichia coli, mutations affecting stem-loop stability resulted in toxicity in this host, presumably due to increased translation.