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research-article

Regulatory interactions between RepA, an essential replication protein, and the DNA repeats of RepFIB from plasmid P307.

Centre for Gene Technology, University of Auckland, New Zealand.

ABSTRACT

The control of RepFIB replication appears to rely on the interaction between an initiator protein (RepA) and two sets of DNA repeat elements located on either side of the repA gene (BCDD'D" and EFGHIJ). In vivo genetic tests demonstrate that the BCDD'D" repeats form part of the origin of replication, while some of the downstream repeat elements (HIJ) are involved in the sensing and setting of plasmid copy number. RepA DNA binding to these groups of repeats has been investigated in vivo by utilizing the fact that the replicon contains three active promoters (orip, repAp, and EFp), one of which has previously been shown to control the expression of repA (repAp). All three promoters are closely associated with the repeat elements flanking repA, and an investigation using lacZ or cml gene fusions has demonstrated that RepA expressed in trans is able to repress each promoter. However, these assays suggest that the transcriptional responses of orip and repAp to RepA repression are significantly different, despite the fact that both promoters are embedded within the BCDD'D" repeat elements. Extra copies of the BCDD'D" or EFG repeats in trans have no effect on RepA repression of repAp embedded in a second copy of the BCDD'D" repeats, but copies of the HIJ or EFGHIJ repeats are able to derepress repAp, suggesting that there is a fundamental difference between RepA-BCDD'D" or -HIJ complexes and RepA-EFG or -EFGHIJ complexes.

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