Nucleic Acids Research Advance Access originally published online on August 9, 2007
Nucleic Acids Research 2007 35(16):5351-5359; doi:10.1093/nar/gkm589
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Nucleic Acids Research, 2007, Vol. 35, No. 16 5351-5359
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
A persistent RNA·DNA hybrid formed by transcription of the Friedreich ataxia triplet repeat in live bacteria, and by T7 RNAP in vitro
Department of Genetics, Louisiana State University Health Sciences Center, 533 Bolivar Street, New Orleans, LA 70112, USA
*To whom correspondence should be addressed. Tel: +1 504 568 6154; Fax: +1 504 568 8500; Email: egrabc{at}lsuhsc.edu
Received November 30, 2006. Revised June 27, 2007. Accepted July 16, 2007.
Expansion of an unstable GAA·TTC repeat in the first intron of the FXN gene causes Friedreich ataxia by reducing frataxin expression. Deficiency of frataxin, an essential mitochondrial protein, leads to progressive neurodegeneration and cardiomyopathy. The degree of frataxin reduction correlates with GAA·TTC tract length, but the mechanism of reduction remains controversial. Here we show that transcription causes extensive RNA·DNA hybrid formation on GAA·TTC templates in bacteria as well as in defined transcription reactions using T7 RNA polymerase in vitro. RNA·DNA hybrids can also form to a lesser extent on smaller, so-called ‘pre-mutation’ size GAA·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·TTC tract and an extensive RNA·DNA hybrid is tightly linked to this arrest. RNA·DNA hybrid formation appears to be an intrinsic property of transcription through long GAA·TTC tracts. RNA·DNA hybrids have a potential role in GAA·TTC tract instability and in the mechanism underlying reduced frataxin mRNA levels in Friedreich Ataxia.
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