An unusually long-lived antisense RNA in plasmid copy number control: in vivo RNAs encoded by the streptococcal plasmid pIP501

Abstract

The main regulator of pIP501 replication is an antisense RNA (RNAIII) that induces transcriptional attenuation of the essential RNAII. Previous studies identified the termination point in vivo and demonstrated attenuation in vitro. This in vivo analysis confirms the appearance of attenuated RNAII dependent on RNAIII. Half-lives and intracellular levels of RNAII and RNAIII were determined: in a Bacillus subtilis cell harboring a wild-type pIP501 plasmid, approximately 50 molecules RNAII and 1000 to 2000 molecules of RNAIII were measured, respectively. The half-life of RNAII was in the range of that of other target RNAs, whereas that of RNAIII (approximately 30 minutes) was unusually long, representing a so far unprecedented case of a metabolically stable antisense RNA regulating plasmid copy number. Long antisense RNA half-life is predicted to yield sluggish control and instability of maintenance. We propose a model for how plasmid pIP501 may avoid this problem by using both the repressor CopR and the antisense RNAIII for control. Four stem-loop mutants of RNAII/RNAIII with elevated copy numbers were characterized for in vitro antisense/target RNA binding, RNAIII half-life, incompatibility, and attenuation in vivo. Two classes were found: interaction mutants and half-life mutants. The former suggest a key function for loop LIII of RNAIII as recognition loop in the primary steps of RNAII/RNAIII interaction.