Unusual Guide-binding Pockets in RNA-targeting pAgo Nucleases

Area of Science:

• Molecular Biology
• Biochemistry
• Microbial Genetics

Background:

• Argonaute nucleases are key enzymes in gene regulation and immunity.
• Prokaryotic Argonautes (pAgos) primarily target DNA and are involved in cellular defense.
• Previously identified DNA > RNA pAgos exhibit unusual guide and target specificities.

Purpose of the Study:

• To characterize additional prokaryotic Argonaute nucleases with DNA guide and RNA target specificity.
• To elucidate the structural basis for the unique nucleic acid recognition and cleavage mechanisms.
• To investigate the role of the C-terminal extension in pAgo function.

Main Methods:

• Bioinformatic analysis of pAgo sequences.
• Biochemical assays for nucleic acid binding and cleavage.
• Site-directed mutagenesis of the C-terminal domain.
• Structural analysis of guide-binding pockets.

Main Results:

• Identified and characterized new DNA > RNA pAgos within existing clades.
• Confirmed non-standard guide-binding pockets in the MID domain.
• Demonstrated that the extended C-terminus, unlike in other pAgos, does not coordinate the guide's 5'-end.
• Showed C-terminus modifications inhibit RNA target cleavage without affecting guide DNA binding.

Conclusions:

• The extended C-terminus in DNA > RNA pAgos plays a crucial role in modulating catalytic activity.
• These findings provide insights into the diverse mechanisms of pAgo function.
• The unique C-terminus presents a potential target for engineering pAgo nucleases.