# Molecular mechanism of the type 2 defense-associated reverse transcriptase

## TL;DR

This study reveals the molecular structure and function of the DRT2 system in bacteria, showing how it uses RNA and a reverse transcriptase to defend against viruses.

## Contribution

The study provides the first structural and biochemical characterization of the DRT2 system's RNA-protein interactions and catalytic mechanism.

## Key findings

- The DRT2 system uses a reverse transcriptase with a 'right-hand' fold and a flexible RNA template for reverse transcription.
- A conserved DDD catalytic triad and a positively charged pocket stabilize the RNA-DNA primer duplex, essential for activity.
- Structural comparisons show DRT2 has unique adaptations for ncRNA binding, highlighting its evolutionary distinctiveness among RT proteins.

## Abstract

Defense-associated reverse transcriptase (DRT) systems play a crucial role in prokaryotic defense mechanisms against phage infections. Among the DRT family, DRT2, DRT3, and DRT9 systems employ protein–noncoding RNA (ncRNA) co-regulatory mechanisms to execute defense functions. Here, we focus on the DRT2 system from Klebsiella pneumoniae, which consists of a reverse transcriptase (RT) and an essential ncRNA component. Using biochemical and structural approaches, we determine the structure of the DRT2 system and reveal detailed interaction modes between the DRT2-RT protein and the ncRNA, especially mediated by specialized anchoring loops and pseudoknot-related structures. The RT protein adopts a conventional “right-hand” fold, while a flexible region of the ncRNA exhibits dynamic conformations, likely serving as the template for reverse transcription. DRT2 mediates reverse transcription through a conserved DDD catalytic triad that coordinates a divalent Mg²⁺ ion. Notably, a short DNA primer-ncRNA duplex is accommodated in a positively charged pocket formed by the thumb and fingers domains, and both interaction analysis and mutagenesis studies confirm that duplex stabilization is essential for activity. Structural comparison and phylogenetic studies of DRT2 and other RT proteins, such as group II introns and UG/Abi RTs, highlight the unique adaptation with a straight extended thumb domain and specialized structures for ncRNA-binding, exemplifying an evolutionary trajectory of RT proteins. In conclusion, our findings expand the understanding of the distinctive characteristics of the DRT2 system and the diversity of prokaryotic antiviral strategies.

Graphical Abstract