UNUSUALLY LONG-LIVED ANTISENSE RNA IN PLASMID COPY NUMBER CONTROL - IN-VIVO RNAS ENCODED BY THE STREPTOCOCCAL PLASMID PIP501

The main regulator of pIP501 replication is an antisense RNA (RNAIII) that induces transcriptional attenuation of the essential RNAII. Previ ous 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 le vels of RNAII and RNAIII were determined: in a Bacillus subtilis cell harboring a wild-type pIP501 plasmid, approximate to 50 molecules RNAI I and 1000 to 2000 molecules of RNAIII were measured, respectively: Th e half-life of RNAII was in the range of that of other target RNAs, wh ereas that of RNAIII (approximate to 30 minutes) was unusually long, r epresenting a so far unprecedented ease of a metabolically stable anti sense 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. Fo ur stem-loop mutants of RNAII/RNAIII with elevated copy numbers were c haracterized for in vitro antisense/target RNA binding, RNAIII half-li fe, incompatibility, and attenuation in vivo. Two classes were found: interaction mutants and half-life mutants. The former suggest a key fu nction for loop LIII of RNAIII as recognition loop in the primary step s of RNAII/RNAIII interaction. (C) 1996 Academic Press Limited