A persistent RNA•DNA hybrid formed by transcription of the Friedreich ataxia triplet repeat in live bacteria, and by T7 RNAP in vitro

Expansion of an unstable GAA center dot TTC reducing frataxin expression. Deficiency of frataxin, an essential mitochondrial protein, leads to progressive neurodegeneration and cardiomyopathy. The degree of frataxin reduction correlates with GAA center dot TTC tract length, but the mechanism of reduction remains controversial. Here we show that transcription causes extensive RNA center dot DNA hybrid formation on GAA center dot TTC templates in bacteria as well as in defined transcription reactions using T7 RNA polymerase in vitro. RNA center dot DNA hybrids can also form to a lesser extent on smaller, so-called 'premutation' size GAA center dot TTC repeats, that do not cause disease, but are prone to expansion. During in vitro transcription of longer repeats, T7 RNA polymerase arrests in the promoter distal end of the GAA center dot TTC tract and an extensive RNA center dot DNA hybrid is tightly linked to this arrest. RNA center dot DNA hybrid formation appears to be an intrinsic property of transcription through long GAA center dot TTC tracts. RNA center dot DNA hybrids have a potential role in GAA center dot TTC tract instability and in the mechanism underlying reduced frataxin mRNA levels in Friedreich Ataxia.