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

A minor population of Escherichia coli contains retroelements called retron s, which encode reverse-transcriptases (RT) to synthesize peculiar satellit e DNAs called multicopy single-stranded DNA (msDNA). These RTs recognize sp ecific RNA structures in their individual primer-template RNAs to initiate cDNA synthesis from the 2'-OH group of a specific internal G residue (branc hing G residue). The resulting products (msDNA) consist of RNA and single-s tranded DNA, sharing hardly any sequence homology. Here, we investigated ho w 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 res idues) and RT-Ec78 (316 residues) share only 71 identical residues (22%), F rom 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 exponentia l enrichment) method with RT-Ec86 and primer RNAs containing random sequenc es, the identical stem-loop structure (including the 3-U loop) to that foun d in the retron-Ec86 primer-template RNA was enriched. In addition, the hig hly conserved 4-base sequence (UAGC), including the branching G residue, wa s 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 seem ingly primitive RNA-dependent DNA polymerases provide insight into the mech anisms for specific protein RNA recognition.