The Involvement of the structures of antisense RNA, target RNA, and their complexes in plasmid R1 copy number regulation

Sammanfattning: Plasmid R1 is a low-copy-number plasmid which belongs to the incompatibility group FII. R1 regulates its own copy number. Initiation of replication of plasmid R1 requires the binding of the RepA protein to oriR1. RepA acts in cis and has to be synthesized de novo for each round of replication. RepA synthesis is negatively regulated by an antisense RNA, CopA, transcribed from a promoter in the opposite direction to that of the repA mRNA. The target of CopA is located in the leader region of the repA mRNA. CopA and CopT interact as folded structnres; the structures of the two RNAs play a crucial role in the control of the translation of repA.There is an upstream open reading frame defined as tap (translational activator peptide) that partially overlaps with the repA gene. Translation of tap was shown to be required for efficient repA expression. Furthermore, translation of tap was inhibited by the binding of CopA to CopT.The aims of this thesis were to investigate why tap translation is required for efficient RepA synthesis, and how CopA inhibits tap and repA translation. Chemical probing experiments and ribosome binding experiments in vitro suggested that the Shine-Dalgarno (SD) sequence and start codon of repA are normally involved in a stable secondary structure which prevents ribosome binding. As tap overlaps with the repA gene, tap translation disruptsthis structure and the repA ribosome binding site (RBS) becomes accessible to the ribosome. In addition, binding of CopA to CopT was shown to inhibit tap translation directly by interfering with ribosome binding to tap RBS.Computer models of the structure of the CopA/CopT complex were generated based on the chemical and the enzymatic probing experiments. These modelling studies suggest that a complete duplex between the two RNAs is not readily formed in vitro. Instead a so-called kissing complex is formed. This complex involves a four-way junction structure and side-by-side helical alignment.