AN RNA PSEUDOKNOT AS THE MOLECULAR SWITCH FOR TRANSLATION OF THE REPZGENE ENCODING THE REPLICATION INITIATOR OF INCI-ALPHA PLASMID COLIB-P9

Translation initiation of the repZ gene encoding the replication initi ator of plasmid ColIb-P9 is not only negatively regulated by the actio n of the antisense Inc RNA encoded in the leader region, but is also c oupled to the translation and termination of a transcribed leader sequ ence, repY, a positive regulatory element for repZ gene expression. Th is translational coupling depends on base pairing between two compleme ntary sequences, 5'-rGGCG-3' and 5'-rCGCC-3', which are located upstre am 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'-r CGCC-3' sequence and the repZ ribosome-binding site. Here we show that the RepZ mRNA leader sequence synthesized in vitro indeed contains se veral stem-loop structures including structures I and III, but not the pseudoknot, However, disruption of structure III, without changing th e repZ ribosome-binding site, by means of base substitution and deleti on 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 observ ed in the absence of Inc RNA. These results strengthen our previously proposed model that the pseudoknot induced by the translation and term ination of the repY reading frame functions as the molecular switch fo r translational initiation of the repZ gene.