CopA and CopT: The Perfect RNA Couple

Abstract: Antisense RNAs regulate gene expression in many bacterial systems. The best characterized examples are from prokaryotic accessory elements such as phages, plasmids and transposons. Many of these antisense RNAs have been identified as plasmid copy number regulators where they regulate the replication frequency of the plasmid by negative feedback. Instability and fast binding kinetics is crucial for the regulatory efficiency of these antisense RNAs. In this thesis, the interaction of the cis-encoded antisense RNA CopA with its target CopT was studied in detail using in vivo reporter gene fusion expression and different in vitro methods, such as surface plasmon resonance, fluorescence resonance energy transfer, and gel-shift assays.Formation of inhibitory complexes differs from simple hybridization reactions between complementary strands. E.g., the binding pathway of CopA and CopT proceeds through a hierarchical order of steps. It initiates by reversible loop-loop contacts, resulting in a helix nucleus of two or three base pairs. This is followed by rapid unidirectional helix progression into the upper stems, resulting in a four-way helical junction structure. It had been suggested that the loop of CopT carries a putative U-turn, a structure first found in tRNA anticodon loops. We showed that this putative U-turn is one of the structural elements of CopA/CopT required to achieve fast binding kinetics. Furthermore, the hypothetical U-turn structure determines the direction of helix progression when the kissing complex progresses to a four-way helical junction structure. Another structural element in CopT is the helical stem adjacent to the recognition loop. This stem is important to present the recognition loop appropriately to provide a scaffold for the U-turn.Furthermore, the role of protein Hfq in the interaction of antisense/target RNA was investigated, since several trans-encoded antisense RNAs had been shown to need this protein to exert their function. In contrast, studies of two cis-encoded antisense RNA systems showed that these antisense RNAs do not rely on Hfq for activity. In this study it was also shown that MicF, a trans-encoded antisense RNA which is dependent on Hfq, is greatly stabilized by this protein.