Published online 16 November 2004
Nucleic Acids Research, Vol. 32 No. 20 © Oxford University Press 2004; all rights reserved
Transient overexpression of mitochondrial transcription factor A (TFAM) is sufficient to stimulate mitochondrial DNA transcription, but not sufficient to increase mtDNA copy number in cultured cells
1 Institute of Vegetative Physiology, Medical Faculty, University of Köln, Robert-Koch-Strasse 39, D-50931 Köln, FRG, 2 Department of Physiology II, University of Heidelberg, Im Neuenheimer Feld 326, D-69120 Heidelberg, FRG and 3 Department of Biochemistry and Molecular and Cellular Biology, University of Zaragoza, Miguel Servet 177, E-50013 Zaragoza, Spain
* To whom correspondence should be addressed. Tel: +49 221 478 3610; Fax: +49 221 478 3538; Email: Rudolf.Wiesner{at}uni-koeln.de; http://www.uni-koeln.de/med-fak/physiologie/index.htm
Present address: Katharina Maniura-Weber, EMPA (Swiss Federal Laboratories for Materials Testing and Research), Lerchenfeldstr. 5, 9014 St Gallen, Switzerland
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
Received August 20, 2004; Revised and Accepted October 14, 2004
Mitochondrial transcription factor A (TFAM) stimulates transcription from mitochondrial DNA (mtDNA) promoters in vitro and in organello. To investigate whether changes of TFAM levels also modulate transcription and replication in situ, the protein was transiently overexpressed in cultured cells. Mitochondrial mRNAs were significantly elevated at early time points, when no expansion of the TFAM pool was yet observed, but were decreased when TFAM levels had doubled, resemb-ling in vitro results. HEK cells contain about 35 molecules of TFAM per mtDNA. High levels of TFAM were not associated with increases of full-length mtDNA, but nucleic acid species sensitive to RNAse H increased. Stimulation of transcription was more evident when TFAM was transiently overexpressed in cells pre-treated with ethidium bromide (EBr) having lowered mtDNA, TFAM and mitochondrial transcript levels. EBr rapidly inhibited mtDNA transcription, while decay of mtDNA was delayed and preferentially slowly migrating, relaxed mtDNA species were depleted. In conclusion, we show that transcription of mtDNA is submaximal in cultured cells and that a subtle increase of TFAM within the matrix is sufficient to stimulate mitochondrial transcription. Thus, this protein meets all criteria for being a key factor regulating mitochondrial transcription in vivo, but other factors are necessary for increasing mtDNA copy number, at least in cultured cells.
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