Non-coding RNA mediates the defense-associated reverse transcriptase (DRT) anti-phage oligomerization transition

Area of Science:

• Molecular Biology
• Microbiology
• Structural Biology

Background:

• Defense-associated reverse transcriptase (DRT) systems are crucial for prokaryotic defense against viral infections.
• The precise molecular mechanisms of DRT systems, particularly their structural dynamics and activation processes, remain largely uncharacterized.
• Understanding these systems is vital for deciphering microbial immunity and developing novel antiviral strategies.

Purpose of the Study:

• To elucidate the molecular mechanisms and structural basis of the two-component DRT9 system's antiviral activity.
• To characterize the role of non-coding RNA (ncRNA) in DRT9 system assembly and activation.
• To investigate the structural transitions and enzymatic activation of DRT9 reverse transcriptase (RT) upon substrate binding.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to determine high-resolution structures of DRT9 in various functional states.
• Biochemical assays to analyze protein-primed DNA synthesis activity and substrate binding.
• Phylogenetic analysis and functional assays to identify key protein domains and their roles.

Main Results:

• The DRT9 system comprises a reverse transcriptase (RT) and a non-coding RNA (ncRNA), exhibiting protein-primed DNA synthesis upon phage infection.
• Cryo-EM structures revealed DRT9 RT forms a dimer of dimers with ncRNA, transitioning to a trimer of dimers upon substrate binding.
• ncRNA facilitates cooperative self-assembly, and substrate binding induces conformational changes via a "lock-switch" mechanism for enzymatic activation.
• A unique N-terminal helix extension was identified as essential for ncRNA stabilization and enzymatic activity, distinguishing DRT9 from other RT systems.

Conclusions:

• The DRT9 system employs a unique ncRNA-mediated oligomerization and conformational switching mechanism for antiviral defense.
• The identified N-terminal helix extension is critical for DRT9 function, highlighting novel aspects of reverse transcriptase activity.
• This study expands the known functional and mechanistic diversity of DRT systems, offering insights into microbial antiviral strategies.