Retron Eco2 breaks tRNAs for antiphage defense

Abstract Retrons are prokaryotic reverse transcriptase systems that produce multicopy single-stranded DNA (msDNA), yet the principles by which they mediate antiviral defense remain largely unresolved. Here, we dissect the mechanism of Eco2, a minimal retron composed of a single reverse transcriptase–nuclease fusion protein. Cryogenic electron microscopy and hydrogen-deuterium exchange mass spectrometry reveal the structures and dynamics of a trimeric nucleoprotein complex assembled within a branched msDNA scaffold, which cages the TOPRIM nuclease. We show that the phage-encoded endonuclease DenB initiates msDNA degradation, thereby unblocking the nuclease active site. Activated Eco2 cuts tRNAs, resulting in translational shutdown for antiphage defense. We further identify ribosomal protein S1 as a putative RNA chaperone that associates with the msDNA precursor. These findings provide insights into the molecular mechanisms of minimal retrons and establish a structural basis for the engineering of Eco2.