Huge thanks to the Wiedenheft Lab—especially Senuri, Murat, Quynh, Royce, Adelaide, Hannah, Adelaide, Ava and our newest faculty Steve—this work wouldn’t have been possible without your support, insight, and suggestions! 🙏

Huge thanks to my co-first author and colleague Nate Burman, who helped me learn cryo-EM. Here’s a fantastic movie he made that captures the key mechanistic steps of this unusual immune system in action!👇

Altogether, our findings reveal how an RT-ATPase immune system assembles a viral surveillance complex using a cDNA ‘harpoon’. Phage flap nucleases trigger its activation, leading to tRNA depletion and translation arrest—while phages fight back by encoding their own tRNAs.

Interestingly phages evade this ATPase-associated RT immune system by encoding their own tRNA-Ser genes! Comparison between phage encoded tRNA-Ser and E.coli tRNA-Ser revealed major differences clustered in the D-loop, which is key for recognition by aminoacyl tRNA synthetase.

Upon co-expressing the retron with phage flap nuclease, we observed nucleoid compaction in E. coli—a hallmark of translation arrest—driven by HNH-mediated depletion of tRNA-Ser. Recently Azam et al. (Nov 24) showed that expression of ATPase+HNH from Eco7 retron depletes tRNA-Tyr.

We then asked: what triggers this system? By sequencing phages that survive retron defense and expressing candidate genes, we found that phage-encoded flap nucleases (yes! the ones that remove Okazaki fragments) are baited to cleave the cDNA scaffold, activating the complex.

We find that HNH is recruited asymmetrically, either in an up or down orientation relative to the RT, and is anchored by a specialized C-terminal claw formed by the ATPase homodimer. Mutations at the ATPase–HNH interface or in claw-stabilizing residues abolished defense.

The long coiled-coil domains of SMC-family ATPases—which typically wrap DNA in repair complexes like Rad50—facilitate interdimer contacts in the retron complex, forming 'bear hug' and 'dorsal fin'-like structures that flank either end of the cDNA scaffold.

Using cryo-EM, we determined that this immune system forms a 364 kDa phage surveillance complex where the extrachromosomal cDNA—made by the RT using the ncRNA—acts as a molecular scaffold to recruit two ATPase homodimers and the nuclease.

Prokaryotic RTs have recently been shown to be key players in antiviral defense. Our story began with a simple question—how does a molecularly odd association of an RT, SMC-ATPase, structured ncRNA, and HNH nuclease (also called retron I-A) orchestrate phage defense?