Abstact

Defense-associated reverse transcriptase (DRT) systems are implicated in prokaryotic resistance to viral infections, yet the molecular mechanisms underlying their functionality remain largely unknown. Here, we characterize a two-component DRT9 system, composed of a reverse transcriptase (RT) and a non-coding RNA (ncRNA), which exhibits a protein-primed DNA synthesis activity upon phage infection. We also determine its cryo-electron microscopy (cryo-EM) structures in different functional states. DRT9 RT binds to ncRNA, forming a dimer of dimers configuration that assembles into a trimer of dimers upon substrate binding. This oligomerization transition, crucial for DRT9-mediated anti-phage defense, is facilitated by a ncRNA cooperative self-assembly manner. Furthermore, substrate binding induces large conformational movements around the catalytic pocket of DRT9 RT, revealing a "lock-switch" mechanism for enzymatic activation. Notably, phylogenetic analysis and functional assays identify a unique N-terminal helix extension required for ncRNA stabilization and enzymatic activity, distinct from previously reported reverse transcriptase systems. Overall, our findings illuminate the molecular basis of DRT9-mediated antiviral defense and expand the functional and mechanistic diversity of the DRT family.