Translationally repressive RNA structures monitored in vivo using temperate DNA bacteriophages

The RNA challenge phage system enables genetic selection of proteins with RNA-binding activity in bacteria. These phages are modified versions of the temperate DNA bacteriophage P22 in which post-transcriptional regulatory events control the developmental fate of the phage. The system was originally developed to identify novel RNA ligands that display reduced affinity for the R17/MS2 coat protein, as well as to select for suppressor coat proteins that recognize mutant RNA ligands. During the course of evaluating whether the HIV-1 Rev protein could direct lysogen development for bacteriophage derivatives that encode Rev response element (RRE) RNA sequences, two examples of RRE RNA ligands that interfere with challenge phage development were identified. In the phage examples described, RRE RNA secondary structure prevents Ant protein biosynthesis and lytic development. Phage lysogen formation occurs efficiently in recipient cells, independent of the expression status of the Rev protein or trans-acting competitor RRE RNA ligands. These studies provide the first example whereby RNA challenge phages may be applied to study RNA folding events and RNA structural interactions in an in vivo context.