The roles of SbcCD and RNaseE in the transcription of GAA·TTC repeats in Escherichia coli

Expansion of GAA·TTC repeats in FXN gene is associated with decreased frataxin production in Frederich's ataxia patients. To study this effect, we have engineered a set of GAA·TTC repeats in the EcoRI site of lacZ gene of plasmid pUC18 as part of the transcription template of the lacZ gene, while keeping its ORF unchanged. The effects of the GAA·TTC repeats on the lacZ expression were investigated in Escherichia coli JM83 and its mutants deficiency in RNA processing, homologous recombination and DNA repair. We found that transcriptions of the GAA strand with different sizes and organizations displayed normal α-complementation when RNase E was functional. By contrast, transcriptions of TTC repeats containing more than 13 triplets failed to support α-complementation, showing RNase-independent but length-dependent effects of TTC repeats on lacZ expression. In addition, we also found that functions of SbcCD, a DNA structure specific nuclease, were needed in the RNase E-dependent lacZ expression of the GAA repeats. These suggested that processing of DNA and RNA is essential to the transcription of the repeats-carrying gene in vivo.