DUAL FUNCTION OF THE COPR GENE-PRODUCT OF PLASMID PIP501

Replication of plasmid pIP501 is regulated at a step subsequent to tra nscription initiation by an antisense RNA (RNAIII) and transcriptional ly by a repressor protein, CopR, Previously, it had been shown that Co pR binds to a 44-bp DNA fragment upstream of and overlapping the repR promoter pII, Subsequently, we found that high-copy-number pIP501 deri vatives lacking copR and low-copy-number derivatives containing copR p roduced the same intracellular amounts of RNAIII. This suggested a sec ond, hitherto-unknown function of CopR, In this report, we show that C opR does not affect the half-life of RNAIII, Instead, we demonstrate i n vivo that, in the presence of both pII and pIII, CopR provided in ci s or in trans causes an increase in the intracellular concentration of RNAIII and that this effect is due to the function of the protein rat her than its mRNA. We suggest that, in the absence of CopR, the increa sed (derepressed) RNAII transcription interferes, in cis, with initiat ion of transcription of RNAIII (convergent transcription), resulting i n a lower RNAIII/plasmid ratio, When CopR is present, the pII promoter is repressed to > 90%, so that convergent transcription is mostly abo lished and RNAIII/plasmid ratios are high. The hypothesis that RNAII t ranscription influences promoter pIII through induced changes in DNA s upercoiling is supported by the finding that the gyrase inhibitor novo biocin affects the accumulation of both sense and antisense RNA, The d ual role of CopR in repression of RNAII transcription and in preventio n of convergent transcription is discussed in the context of replicati on control of pIP501.