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

Abstract

A minor population of Escherichia coli contains retro-elements called retrons, which encode reverse transcriptases (RT) to synthesize peculiar satellite DNAs called multicopy single-stranded DNA (msDNA). These RTs recognize specific RNA structures in their individual primer-template RNAs to initiate cDNA synthesis from the 2'-OH group of a specific internal G residue (branching G residue). The resulting products (msDNA) consist of RNA and single-stranded DNA, sharing hardly any sequence homology. Here, we investigated how RT-Ec86 recognizes the specific RNA structure in its primer-template RNA. On the basis of structural comparison with HIV-1 RT, domain exchanges were carried out between two E. coli RTs, RT-Ec86 and RT-Ec73. RT-Ec86 (320 residues) and RT-Ec73 (316 residues) share only 71 identical residues (22%). From the analysis of 10 such constructs, the C-terminal 91-residue sequence of RT-Ec86 was found to be essential for the recognition of the unique stem-loop structure and the branching G residue in the primer-template RNA for retron-Ec86. Using the SELEX (systematic evolution of ligands by exponential enrichment) method with RT-Ec86 and primer RNAs containing random sequences, the identical stem-loop structure (including the 3-U loop) to that found in the retron-Ec86 primer-template RNA was enriched. In addition, the highly conserved 4-base sequence (UAGC), including the branching G residue, was also enriched. These results indicate that the highly diverse C-terminal region recognizes specific stem-loop structures and the branching G residue located upstream of the stem-loop structure. The present results with seemingly primitive RNA-dependent DNA polymerases provide insight into the mechanisms for specific protein RNA recognition.