Abstract 1006 Oligomerization-mediated activation of a short prokaryotic Argonaute

This is the authors' abstract. We don't add key points for this paper.

While eukaryotic Argonautes and long prokaryotic Argonautes (pAgos) cleave nucleic acids, some short pAgos lack nuclease activity and hydrolyze NAD(P)+ to induce bacterial cell death. We present a hierarchical activation pathway for SPARTA, a short pAgo system consisting of an Ago protein and an associated protein TIR-APAZ. SPARTA progresses through distinct oligomeric forms, including a monomeric apo state, a monomeric RNA-DNA-bound state, two dimeric RNA-DNA-bound states, and a tetrameric RNA-DNA-bound active state. These structural snapshots together identify oligomerization as a mechanistic principle of SPARTA activation. Apo SPARTA is inactive, and its RNA-DNA-binding channel is occupied by an auto-inhibitory motif in TIR-APAZ. Upon RNA-DNA binding, SPARTA transitions from a monomer to a symmetric and then asymmetric dimer, in which two TIR domains interact via charge and shape complementarity. Next, two dimers assemble into a tetramer with a central TIR cluster responsible for hydrolyzing NAD(P)+. Additionally, we observed unique features of SPARTA-RNA-DNA interactions, including competition between the DNA 3' end and the auto-inhibitory motif, interactions between the RNA G2 nucleotide and Ago, and splaying of the RNA-DNA duplex by two loops exclusive to short pAgos. Together, by using cryo-EM technique and biochemical experiments, we depict a mechanistic basis for the activation of short pAgos, a large section of the Ago superfamily.