Characterizing retron Efe1 reverse transcriptase interactions with ncRNA

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2024-04

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Retrons are diverse bacterial anti-phage defense systems. A retron operon consists of a reverse transcriptase, an accessory protein, and a structured non-coding RNA that serves as the primer and template for reverse transcription. Retrons are currently being developed into new gene editing tools in bacteria, plants, and mammalian cells. A new retron system discovered in the Finkelstein lab, Efe1, has shown higher gene editing rates in mammalian cells than the current standard in retron gene editing, Eco1. Discovering what makes Efe1 better than Eco1 can elucidate the molecular mechanisms behind retron functions. Here, I investigate Efe1 reverse transcriptase and use cryo-electron microscopy to reconstruct a 3.9 Å density map of its RT-msDNA complex. Efe1 complex is highly similar to Eco1 complex, except that it is a monomer and its msDNA has a more rigid DNA stem loop than Eco1. Efe1 reverse transcriptase solubility decreases drastically in the absence of its cognate ncRNA. Efe1 reverse transcriptase can also be solubilized by Eco1 ncRNA and produce Eco1 msDNA. Mutating catalytic residues in Efe1 reverse transcriptase ablates msDNA production and reduces solubility. These findings offer insight into retron reverse transcriptase interactions with ncRNA that dictate proper protein folding and provide some guidance for future attempts at purifying retron reverse transcriptase alone.

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