Copy number control of IncIalpha plasmid ColIb-P9 by competition between pseudoknot formation and antisense RNA binding at a specific RNA site

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Abstract
Replication of a low‐copy‐number IncIα plasmid ColIb‐P9 depends on expression of the repZ gene encoding the replication initiator protein. repZ expression is negatively controlled by the small antisense Inc RNA, and requires formation of a pseudoknot in the RepZ mRNA consisting of stem–loop I, the Inc RNA target, and a downstream sequence complementary to the loop I. The loop I sequence comprises 5′‐rUUGGCG‐3′, conserved in many prokaryotic antisense systems, and was proposed to be the important site of copy number control. Here we show that the level of repZ expression is rate‐limiting for replication and thus copy number, by comparing the levels of repZ expression and copy number from different mutant ColIb‐P9 derivatives defective in Inc RNA and pseudoknot formation. Kinetic analyses using in vitro transcribed RNAs indicate that Inc RNA binding and the pseudoknot formation are competitive at the level of initial base paring to loop I. This initial interaction is stimulated by the presence of the loop U residue in the 5′‐rUUGGCG‐3′ motif. These results indicate that the competition between the two RNA–RNA interactions at the specific site is a novel regulatory mechanism for establishing the constant level of repZ expression and thus copy number.