Dual function of the copR gene product of plasmid pIP501

Replication of plasmid pIP501 is regulated at a step subsequent to transcription initiation by an antisense RNA (RNAIII) and transcriptionally by a repressor protein, CopR, Previously, it had been shown that CopR binds to a 44-bp DNA fragment upstream of and overlapping the repR promoter pII, Subsequently, we found that high-copy-number pIP501 derivatives lacking copR and low-copy-number derivatives containing copR produced the same intracellular amounts of RNAIII. This suggested a second, hitherto-unknown function of CopR, In this report, we show that CopR does not affect the half-life of RNAIII, Instead, we demonstrate in vivo that, in the presence of both pII and pIII, CopR provided in cis or in trans causes an increase in the intracellular concentration of RNAIII and that this effect is due to the function of the protein rather than its mRNA. We suggest that, in the absence of CopR, the increased (derepressed) RNAII transcription interferes, in cis, with initiation of transcription of RNAIII (convergent transcription), resulting in a lower RNAIII/plasmid ratio, When CopR is present, the pII promoter is repressed to > 90%, so that convergent transcription is mostly abolished and RNAIII/plasmid ratios are high. The hypothesis that RNAII transcription influences promoter pIII through induced changes in DNA supercoiling is supported by the finding that the gyrase inhibitor novobiocin affects the accumulation of both sense and antisense RNA, The dual role of CopR in repression of RNAII transcription and in prevention of convergent transcription is discussed in the context of replication control of pIP501.