An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Group II introns and non-LTR retrotransposons utilize highly processive reverse transcriptases (RTs).
• These retroelement RTs are structurally distinct from retroviral RTs used in biotechnology.
• Quantitative analysis of retroelement RT processivity, efficiency, and accuracy is limited.

Purpose of the Study:

• To characterize the processivity of a group II intron maturase RT from Eubacterium rectale (E.r).
• To provide a structure-guided analysis of E.r. maturase RT (MarathonRT) capabilities.
• To explore engineering strategies for enhancing RT performance.

Main Methods:

• Processivity assays were performed on MarathonRT.
• Structural analysis identified key features like the α-loop.
• Single-molecule sequencing was used to assess error frequency.
• Primer utilization efficiency was evaluated before and after enzyme reengineering.

Main Results:

• MarathonRT demonstrates high processivity, copying transcripts of at least 10 kb.
• Its intrinsic processivity surpasses commercial enzymes like Superscript IV (SSIV).
• An α-loop in the finger subdomain contributes to enhanced processivity.
• A positively charged surface RNA binding site negatively impacts primer utilization efficiency.
• Reengineering this site improves MarathonRT capabilities.
• MarathonRT exhibits error frequencies comparable to high-performance RTs like SSIV.

Conclusions:

• Retroelement RTs possess inherent structural features for enhanced processivity.
• MarathonRT serves as a model for understanding and engineering these enzymes.
• This work lays the foundation for developing novel RT tools for biotechnology and research.