The plasmid ColIb-P9 antisense Inc RNA controls expression of the RepZ replication protein and its positive regulator repY with different mechanisms.

The autonomous replication region of plasmid ColIb-P9 contains repZ encoding the RepZ replication protein, and inc and repY as the negative and positive regulators of repZ translation, respectively. inc encodes the antisense Inc RNA, and repY is a short open reading frame upstream of repZ. Translation of repY enables repZ translation by inducing formation of a pseudoknot containing stem-loop I, which base pairs with the sequence preceding the repZ start codon. Inc RNA inhibits both repY translation and formation of the pseudoknot by binding to the loop I. To investigate control of repY expression by Inc RNA, we isolated a number of mutations that express repY in the presence of Inc RNA. One class of mutations delete a part of another stem-loop (II), which derepresses repY expression by initiating translation at codon 10 (GUG), located within this structure. Point mutations in stem-loop II can also derepress repY translation, and the introduction of compensatory base-changes restores control of repY translation. These results not only indicate that suppressing a cryptic start codon by secondary structure is important for maintaining the translational control of repZ but also demonstrate that the position of start site for repY translation is critical for its control by Inc RNA. Thus, Inc RNA controls repY translation by binding in the vicinity of the start codon, in contrast to the control of repZ expression at the level of loop-loop interaction.