The GAA•TTC triplet repeat expanded in Friedreich’s ataxia impedes transcription elongation by T7 RNA polymerase in a length and supercoil dependent manner

Large expansions of the trinucleotide repeat GAA•TTC within the first intron of the X25 (frataxin) gene cause Friedreich’s ataxia, the most common inherited ataxia. Expansion leads to reduced levels of frataxin mRNA in affected individuals. Here we show that GAA•TTC tracts, in the absence of any other frataxin gene sequences, can reduce the amount of GAA-containing transcript produced in a defined in vitro transcription system. This effect is due to an impediment to elongation that forms in the GAA•TTC tract during transcription, a phenomenon that is exacerbated by both superhelical stress and increased tract length. On supercoiled templates the major truncations of the GAA-containing transcripts occur in the distal (3′) end of the GAA repeat. To account for these observations we present a model in which an RNA polymerase advancing within a long GAA•TTC tract initiates the transient formation of an R•R•Y intramolecular DNA triplex. The non-template (GAA) strand folds back creating a loop in the template strand, and the polymerase is paused at the distal triplex–duplex junction.