Highly specific recognition of primer RNA structures for 2'-OH priming reaction by bacterial reverse transcriptases

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Escherichia coli retrons encode reverse transcriptases (RTs) that synthesize multicopy single-stranded DNA (msDNA).
• These RTs initiate DNA synthesis by recognizing specific RNA structures and a branching G residue on primer-template RNAs.
• The msDNA products comprise RNA and single-stranded DNA with minimal sequence homology.

Purpose of the Study:

• To investigate the mechanism by which RT-Ec86 recognizes specific RNA structures and the branching G residue.
• To identify the specific protein domains responsible for primer-template RNA recognition in retron-Ec86.

Main Methods:

• Structural comparison and domain exchange experiments between two E. coli RTs (RT-Ec86 and RT-Ec73).
• Systematic evolution of ligands by exponential enrichment (SELEX) using RT-Ec86 and random-sequence primer RNAs.

Main Results:

• The C-terminal 91-residue sequence of RT-Ec86 was identified as essential for recognizing the stem-loop structure and branching G residue.
• SELEX enriched for a stem-loop structure (including a 3-U loop) and a conserved UAGC sequence containing the branching G residue.
• These findings indicate that the C-terminal region of RT-Ec86 mediates specific recognition of RNA secondary structures and the branching G site.

Conclusions:

• The C-terminal region of RT-Ec86 plays a crucial role in the specific recognition of primer-template RNA structures.
• This study provides insights into the mechanisms of specific protein-RNA recognition in primitive RNA-dependent DNA polymerases.