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.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.
Grabczyk
Ed
E
Department of Genetics, Louisiana State University Health Sciences Center, 533 Bolivar Street, New Orleans, LA 70112, USA. egrabc@lsuhsc.edu
Mancuso
Miriam
M
Sammarco
Mimi C
MC
D001419:Bacteria / Q000235:genetics; D004247:DNA / Q000096:biosynthesis*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D033862:Iron-Binding Proteins / Q000235:genetics*; D012313:RNA / Q000096:biosynthesis*; D013698:Templates, Genetic; D014158:Transcription, Genetic; D018911:Trinucleotide Repeats; D014764:Viral Proteins / Q000378:metabolism*; D000096923:Frataxin
D004247:DNA; D012321:DNA-Directed RNA Polymerases; D033862:Iron-Binding Proteins; D012313:RNA; D014764:Viral Proteins; D000096923:Frataxin; C062210:bacteriophage T7 RNA polymerase
J Mol Biol. 1999 Nov 26;294(2):321-32
10610761
Nucleic Acids Res. 2000 Dec 15;28(24):4930-7
11121484
Nucleic Acids Res. 2000 Jul 15;28(14):2815-22
10908340
Nat Genet. 2001 Apr;27(4):407-11
11279522
Brain Res Bull. 2001 Oct-Nov 1;56(3-4):367-73
11719274
Hum Mol Genet. 2002 Jan 15;11(2):191-8
11809728
Nature. 2002 May 16;417(6886):304-8
11979277
Nature. 2003 Apr 24;422(6934):909-13
12712207
Nat Immunol. 2003 May;4(5):442-51
12679812
J Biol Chem. 2003 Oct 3;278(40):39037-43
12871933
Mol Cell. 2003 Sep;12(3):711-21
14527416
Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):1858-62
14769921
Genes Dev. 2004 Jul 1;18(13):1618-29
15231739
EMBO J. 2004 Jul 7;23(13):2620-31
15192704
Mol Cell. 2004 Sep 10;15(5):777-88
15350221
Proc Natl Acad Sci U S A. 1972 Jul;69(7):1863-7
4558661
Anal Biochem. 1987 Apr;162(1):156-9
2440339
Proc Natl Acad Sci U S A. 1987 Oct;84(20):7024-7
2823250
Cell. 1988 Jul 29;54(3):403-11
2840207
J Biol Chem. 1989 Apr 5;264(10):5944-9
2647730
Nature. 1990 Nov 22;348(6299):342-4
1701219
New Biol. 1990 Sep;2(9):771-7
2177653
Nucleic Acids Res. 1993 Feb 11;21(3):365-72
8441648
Biochemistry. 1994 May 3;33(17):5298-304
7513557
J Biol Chem. 1995 Jan 27;270(4):1791-7
7829515
Biochemistry. 1995 Mar 28;34(12):4125-32
7535100
J Bacteriol. 1995 Jul;177(14):4121-30
7608087
Biochemistry. 1995 Aug 29;34(34):10807-15
7662660
Annu Rev Biochem. 1995;64:65-95
7574496
Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12250-4
8618879
Nucleic Acids Res. 1995 Dec 25;23(24):5006-11
8559658
Science. 1996 Mar 8;271(5254):1423-7
8596916
Nucleic Acids Res. 1996 Jun 1;24(11):1992-8
8668527
Am J Hum Genet. 1996 Sep;59(3):554-60
8751856
Annu Rev Biochem. 1996;65:635-92
8811192
Ann Neurol. 1997 May;41(5):675-82
9153531
Am J Hum Genet. 1997 Jul;61(1):101-10
9245990
Neurology. 1997 Aug;49(2):606-10
9270608
Am J Hum Genet. 1998 Jan;62(1):111-21
9443873
J Biol Chem. 1998 Jun 5;273(23):14588-95
9603975
Mol Cell. 1998 Mar;1(4):583-93
9660942
Hum Mol Genet. 1998 Nov;7(12):1901-6
9811933
Anal Biochem. 1999 Feb 1;267(1):241-3
9918680
Mol Cell. 1999 Apr;3(4):465-75
10230399
J Biol Chem. 1999 Jun 4;274(23):16659-64
10347234
Mol Cell. 2005 Jun 10;18(6):711-22
15949445
Genes Dev. 2005 Jul 1;19(13):1512-7
15998806
Cell. 2005 Aug 12;122(3):365-78
16096057
Proc Natl Acad Sci U S A. 2006 Mar 28;103(13):5030-5
16547142
J Mol Biol. 2006 Jul 21;360(4):800-13
16784751
J Mol Biol. 2006 Aug 25;361(4):634-43
16781733
J Mol Biol. 2006 Aug 25;361(4):644-58
16876197
Nat Chem Biol. 2006 Oct;2(10):551-8
16921367
J Mol Biol. 2000 Jun 2;299(2):325-35
10860741