Reverse transcriptases from bacterial retrons require specific secondary structures at the 5'-end of the template for the cDNA priming reaction

Abstract

Multicopy single-stranded DNA (msDNA) is a peculiar molecule consisting of a single-stranded DNA that is branched out from an internal G residue of an RNA molecule (msdRNA) via a 2',5'-phosphodiester linkage. The genetic unit required for msDNA synthesis is designated "retron" and consists of msr (a gene for msdRNA), msd (a gene for msDNA), and a gene for reverse transcriptase (RT) in a single operon. To date, four different msDNAs have been isolated from Escherichia coli. They do not share any primary sequences in either RNA or DNA. To elucidate the specificity of bacterial RT for msDNA synthesis, the msr-msd region from retron-Ec67 was introduced into E. coli cells producing RT-Ec73, or the msr-msd region from retron-Ec73 into E. coli cells producing RT-Ec67. In both cases, msDNA was not synthesized. However, when the msdRNA coding regions (msr) for retron-Ec67 and -Ec73 were mutually exchanged and the chimeric genes were introduced into E. coli cells producing either RT-Ec67 or RT-Ec73, it was thus found that msDNA was produced only when msr and RT were from the same retron. Requirement of the msr region for msDNA synthesis by RT was further investigated by mutations in the msr region for retron-Ec67. These analyses revealed that there is a strict requirement for specific primary sequences as well as the secondary structure in msdRNA. This finding is discussed in relationship to the mechanism of the priming reaction of cDNA synthesis by eukaryotic retroviral RTs using tRNAs.