An RNA Pseudoknot as the Molecular Switch for Translation of therepZ Gene Encoding the Replication Initiator of IncIα Plasmid ColIb-P9

Translation initiation of the repZ gene encoding the replication initiator of plasmid ColIb-P9 is not only negatively regulated by the action of the antisense Inc RNA encoded in the leader region, but is also coupled to the translation and termination of a transcribed leader sequence, repY , a positive regulatory element for repZ gene expression. This translational coupling depends on base pairing between two complementary sequences, 5′-rGGCG-3′ and 5′-rCGCC-3′, which are located upstream of and in the middle of repY , respectively, and have the potential to form a pseudoknot with the stem-loop structure I. Another stem-loop called structure III near the 3′-end of repY sequesters both the 5′-rCGCC-3′ sequence and the repZ ribosome-binding site. Here we show that the RepZ mRNA leader sequence synthesized in vitro indeed contains several stem-loop structures including structures I and III, but not the pseudoknot. However, disruption of structure III, without changing the repZ ribosome-binding site, by means of base substitution and deletion induces base pairing between the two short complementary sequences distantly separated, resulting in the formation of a pseudoknot. When the pseudoknot is allowed to form in vivo due to the same mutations, a maximum level of repZ expression is obtained comparable to one observed in the absence of Inc RNA. These results strengthen our previously proposed model that the pseudoknot induced by the translation and termination of the repY reading frame functions as the molecular switch for translational initiation of the repZ gene.