Architecture and mechanism of a dual-enzyme retron system in prokaryotic immunity.

Retrons are bacterial genetic retroelements encoding a reverse transcriptase (RT) and a non-coding RNA (ncRNA)-multi-copy single-stranded DNA (msDNA) hybrid. Diverse effector proteins or domains are found to associate with retrons, typically forming tripartite toxin-antitoxin systems involved in anti-phage defense. Although retrons have attracted growing interest in genome editing technologies, the mechanisms underlying most retron-mediated immune systems remain poorly understood. Here, we characterize a distinct quaternary retron system, Ec78, harboring a dual-component effector complex in which the PtuA ATPase and PtuB nuclease act in concert to mediate phage clearance. The cryo-EM structure of the Ec78 complex adopts a flower-basket-like architecture, with two Ec78 retrons engaging the PtuAB effector complexes through a msDNA-insertion assembly mechanism. Shortening of msDNA in length releases the PtuAB from Ec78 retron and triggers its activation. The cryo-EM structure of the retron-unbound effector complex further reveals an arginine-lysine finger loop on the PtuB nuclease that undergoes an ordered-to-disordered transition during enzymatic activation. These findings delineate the molecular basis underlying the Ec78 system in antiviral defense and highlight the mechanistic diversity of retron systems in prokaryotic immunity.