Structural basis for template switching by a group II intron-encoded non-LTR-retroelement reverse transcriptase.

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Reverse transcriptases (RTs) are enzymes capable of template switching (TS), a process vital for retroviral replication, recombination, and RNA sequencing (RNA-Seq) adapter addition.
• TS enables the joining of discontinuous nucleic acid sequences, potentially enhancing retroelement fitness and facilitating DNA synthesis on damaged templates.

Purpose of the Study:

• To determine the X-ray crystal structure of a template switching (TS) complex formed by a group II intron RT.
• To elucidate the structural mechanisms underlying TS and nontemplated nucleotide addition by RTs.
• To identify potential strategies for improving TS efficiency in RNA-Seq applications.

Main Methods:

• X-ray crystallography was employed to determine the structure of a group II intron RT in a TS complex.
• The complex involved an acceptor RNA, a donor RNA template-DNA primer heteroduplex, and a 1-nt 3'-DNA overhang.
• Structure-guided mutagenesis was performed to identify key amino acids involved in acceptor RNA binding and nucleotide addition.

Main Results:

• The crystal structure revealed the binding of the acceptor RNA's 3' end in a specific pocket formed by an N-terminal extension and the RT fingertips loop.
• The 3' nucleotide of the acceptor RNA was observed to base-pair with the 3'-DNA overhang, and the penultimate nucleotide with the incoming dNTP.
• A phenylalanine residue near the active site was identified as crucial for nontemplated nucleotide addition, and its mutation reduced multiple sequential template switches in RNA-Seq.

Conclusions:

• The study provides novel structural insights into the mechanism of template switching and nontemplated nucleotide addition by reverse transcriptases.
• The findings suggest common structural features for TS in non-long terminal repeat retroelement RTs and viral RNA-dependent RNA polymerases.
• The identified structural elements and residues offer targets for optimizing RT activity for enhanced RNA sequencing protocols.