In prokaryotes, reverse transcriptases (RTs) — which catalyse the transcription of RNA into DNA — serve important roles in genomic mobility, adaptation and evolution. Several RTs have been associated with phage defence systems, of which a subgroup was found to require the presence of a specific non-coding RNA (ncRNA). A study in Science looking into the defence mechanism of defence-associated RTs of the type 2 class (called ‘DRT2’ here) finds a novel mode of gene regulation whereby DRT2s facilitate the assembly of a novel, toxin-encoding gene from a ncRNA template.

To test whether the RT component of DRT2 was reverse transcribing its ncRNA component after phage infection, the authors infected DRT2-expressing E. coli with T5 phages, extracted DNA by alkaline lysis to deplete genomic DNA, and sequenced all double-stranded DNA. The resulting read coverage and structure suggested that a 120-bp central region of the ncDNA was reverse transcribed into a repetitive cDNA, with at least some of the cDNA in infected cells being double stranded. Long-read sequencing of total DNA from DRT2-expressing E. coli after T5 infection confirmed the presence of reads containing varying numbers of tandem repeats of the 120-bp region. Tiled transversion mutations across the template region abolished phage defence but not the generation of concatemeric cDNA, which suggested that the cDNA sequence itself was pivotal for the defence function.