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 characterized a distinct quaternary retron system, Ec78, harboring a dual-enzymatic 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 unique msDNA-insertion assembly mechanism. Interestingly, a sensing loop on the RT protein tightly monitors the length of the msDNA, which is likely responsible for phage detection and the subsequent release of the toxic effector complex. We further determined the cryo-EM structure of the retron-unbound effector complex, revealing an arginine-lysine finger loop on the PtuB nuclease that undergoes an ordered-to-disordered transition for enzymatic activation. Together, our work not only delineates the molecular basis underlying the Ec78 system in antiviral defense but also highlights the mechanistic diversity of retron systems in prokaryotic immunity.