Crystal structures of restrictocin-inhibitor complexes with implications for RNA recognition and base flipping

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• The cytotoxin sarcin inhibits protein synthesis by cleaving a specific phosphodiester bond in ribosomes.
• Understanding the molecular mechanisms of toxin-ribosome interaction is crucial for deciphering protein synthesis regulation.
• The sarcin/ricin loop (SRL) of the 28S rRNA is the specific target for these toxins.

Purpose of the Study:

• To elucidate the molecular basis of how the sarcin homolog restrictocin recognizes and cleaves the SRL RNA.
• To visualize the structural interactions between restrictocin and SRL RNA analogs.
• To provide insights into the mechanism of site-specific RNA cleavage by protein endonucleases.

Main Methods:

• Cocrystallography was employed to determine the structures of restrictocin bound to SRL RNA analogs.
• Analysis of three distinct cocrystal structures provided detailed views of toxin-RNA interactions.
• Structural comparisons were used to understand target site recognition and cleavage mechanisms.

Main Results:

• Restrictocin binds to the bulged-G motif and an unfolded tetraloop of the SRL RNA.
• Specific toxin loops mediate target site selection by interacting with key recognition bases (e.g., G4319) and the RNA backbone.
• Base flipping of the tetraloop positions the target nucleotide for efficient cleavage, with the nucleophile nearly aligned for attack.

Conclusions:

• These structures offer the first detailed visualization of a site-specific protein endonuclease interacting with a folded RNA substrate.
• The findings reveal a sophisticated mechanism for RNA recognition and cleavage involving base flipping and precise nucleophile positioning.
• This study provides fundamental insights into the molecular interactions governing rRNA cleavage by cytotoxins, impacting our understanding of protein synthesis inhibition.