Dual function of the copR gene product of plasmid pIP501

Abstract

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.Replication of the broad-host-range plasmid pIP501 (19) is controlled at a step subsequent to transcription initiation by CopR (7) and posttranscriptionally by an antisense RNA (RNAIII) (11). RNAIII induces transcriptional attenuation of RNAII within the leader region of the repR mRNA (11,12). CopR binds to a 44-bp sequence upstream of and partially overlapping the repR promoter pII, thereby repressing RNAII synthesis about 10-fold (7). Deletion of copR resulted in a 10-to 20-fold increase of pIP501 copy number (9), consistent with the elevated synthesis of RNAII, which encodes the rate-limiting RepR protein (10). Recently, we measured the steady-state concentrations of RNAIII in Bacillus subtilis cells in high-copynumber and low-copy-number pIP501 derivatives. Since RNAIII is synthesized constitutively, its concentration should be directly correlated with plasmid copy number. Surprisingly, we found that this was not the case: both pPR1 (rnaIII ϩ copR; 50 to 100 copies/cell) and pCOP4 (rnaIII ϩ copR ϩ ; 5 to 10 copies/ cell) expressed about 1,000 to 2,000 molecules of RNAIII/cell (1.4 to 2.8 M [13]). This suggested that, in addition to its function as a repressor of RNAII transcription, CopR is required for RNAIII accumulation. Previous analyses using transcriptional promoter-lacZ fusions ruled out the idea that CopR activates transcription from pIII (7). Hence, CopR must act either by affecting the stability of R...

Transcriptional interference – a crash course

“…Examples of TI as a means of gene regulation are common in the genomes of 'extrachromosomal' elements such as bacteriophages [19,[36][37][38], transposable elements [39], insertion sequences [40] and plasmids [25,26]. Perhaps the highly compact nature of these genomes favours the evolution of convergent transcription to regulate gene expression.…”

“…In addition to these examples, TI is exploited in the regulation of some cellular activities, such as the initiation of plasmid replication [25,26]. There is also the potential of TI to: (i) contribute to the overall interference accorded to RNAi, caused by the presence of in cis intergenic convergent promoters [13,27,28]; and (ii) provide a different gene product, by alternative gene splicing from the extended transcript, following the interference of a downstream promoter by the activity of an upstream in-tandem promoter [29].…”

“…In the mouse, deletion of the promoter of unr leads to elevated transcription of N-ras and embryonic lethality for the homozygous mutant. An analogous situation applies to the defect in expression of the human α-globin genes in α-thalassemia, as a result of mutational read-through from the upstream promoter of the α2-globin gene [24].In addition to these examples, TI is exploited in the regulation of some cellular activities, such as the initiation of plasmid replication [25,26]. There is also the potential of TI to: (i) contribute to the overall interference accorded to RNAi, caused by the presence of in cis intergenic convergent promoters [13,27,28]; and (ii) provide a different gene product, by alternative gene splicing from the extended transcript, following the interference of a downstream promoter by the activity of an upstream in-tandem promoter [29].…”

Transcriptional repressor CopR acts by inhibiting RNA polymerase binding

“…B. subtilis strain 168 containing an rpoC gene with a 39 terminal His 6 -tag was (Brantl & Wagner, 1997). Lower part: schematic representation of the DNA sequence containing the CopR target.…”

Transcriptional Repressor CopR: Use of SELEX To Study the copR Operator Indicates that Evolution Was Directed at Maximal Binding Affinity

“…Whereas RNAIII exerts its inhibitory effect by premature termination (attenuation) of the essential repR mRNA (6,8), CopR has a dual function. On the one hand, it represses transcription from the essential repR promoter pII about 10-to 20-fold (7); on the other hand, it prevents convergent transcription from pII and pIII (antisense promoter), thereby indirectly increasing transcription initiation at pIII (9). Previously, it was found that CopR contacts the DNA asymmetrically at two consecutive major grooves that share the consensus motif 5Ј-CGTG (28).…”