Translationally repressive RNA structures monitored in vivo using temperate DNA bacteriophages

Area of Science:

• Bacteriophage genetics
• Molecular biology
• RNA-protein interactions

Background:

• The RNA challenge phage system, a modification of bacteriophage P22, uses post-transcriptional regulation to control phage development.
• The system was initially designed to identify novel RNA ligands and suppressor proteins.
• Previous applications focused on R17/MS2 coat protein interactions.

Purpose of the Study:

• To evaluate the HIV-1 Rev protein's ability to direct lysogen development in bacteriophages encoding Rev response element (RRE) RNA.
• To identify novel RNA ligands that interact with RRE sequences within the phage system.
• To investigate the in vivo impact of RNA secondary structure on phage development.

Main Methods:

• Genetic engineering of bacteriophage P22 to incorporate RRE RNA sequences.
• Selection of phage derivatives based on developmental outcomes (lytic vs. lysogenic).
• Analysis of RRE RNA structure and its effect on phage gene expression.

Main Results:

• Two RRE RNA ligands were identified that inhibit challenge phage development.
• RRE RNA secondary structure was found to prevent Ant protein biosynthesis and lytic development.
• Phage lysogen formation was efficient and independent of Rev protein expression or competing RRE RNAs.

Conclusions:

• RNA challenge phages can be utilized to study in vivo RNA folding and structural interactions.
• The system provides a novel platform for investigating RNA-mediated regulation in a bacterial context.
• This work expands the application of phage display systems to RNA structural biology.