Using circular permutation analysis to redefine the R17 coat protein binding site

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• The bacteriophage R17 coat protein binds to a specific RNA hairpin structure.
• This RNA structure features a single purine nucleotide bulge within the helical stem.
• Understanding protein-RNA interactions is crucial for molecular biology.

Purpose of the Study:

• To investigate the impact of phosphodiester backbone breaks on R17 coat protein-RNA binding affinity using Circular Permutation Analysis (CPA).
• To precisely map the 5' and 3' boundaries of the R17 coat protein binding site on RNA.
• To compare binding site sizes determined by different experimental methods.

Main Methods:

• Circular Permutation Analysis (CPA) to assess binding affinity after backbone cleavage.
• Synthesis of circularly permuted RNA isomers.
• Terminal truncation experiments to verify binding site boundaries.
• 3-ethyl-1-nitrosourea-modification interference assays.

Main Results:

• Cleavage of most phosphodiester bonds in the RNA hairpin significantly reduced binding affinity, likely due to hairpin destabilization.
• A specific circularly permuted isomer, with ends at the bulged nucleotide, maintained wild-type binding affinity.
• Extending the 5' end of this isomer reduced binding, suggesting limitations for its function within larger RNAs.
• CPA identified a shorter 5' boundary (-15 to +2) compared to previous studies (-17 to +2), confirmed by truncation experiments.
• A minimal binding fragment (-14 to +2) showed tight binding.
• 3-ethyl-1-nitrosourea-modification interference suggested a larger 5' boundary (-16 to +1), potentially due to steric effects.

Conclusions:

• The structural integrity of the RNA hairpin is critical for high-affinity R17 coat protein binding.
• Circular permutation analysis is a valuable tool for probing protein-RNA interaction sites and boundaries.
• The apparent size of a protein's RNA binding site can be method-dependent.