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Functional Analysis of in Vivo and in Organello Footprinting of HeLa Cell Mitochondrial DNA in Relationship to ATP and Ethidium Bromide Effects on Transcription

Micol, Vicente and Fernádez-Silva, Patricio and Attardi, Giuseppe (1997) Functional Analysis of in Vivo and in Organello Footprinting of HeLa Cell Mitochondrial DNA in Relationship to ATP and Ethidium Bromide Effects on Transcription. Journal of Biological Chemistry, 272 (30). pp. 18896-18904. ISSN 0021-9258.

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In vivo and in organello footprinting techniques based on methylation interference have been utilized to investigate protein-DNA interactions in the transcription initiation and rDNA transcription termination regions of human mitochondrial DNA (mtDNA) in functionally active mitochondria. In particular, the changes in methylation reactivity of these regions in response to treatment of the organelles with ATP or ethidium bromide, which affects differentially the rates of mitochondrial rRNA and mRNA synthesis, have been analyzed. Two major sites of protein-DNA interactions have been identified in the main control region of mtDNA, both in vivo and in organello, which correspond to the regions of the light-strand promoter and heavy-strand rRNA-specific promoter. The in organello footprinting of the latter showed ATP- and ethidium bromide-dependent modifications that could be correlated with changes in the rate of rRNA but not of mRNA synthesis. By contrast, no ATP effects were observed on the in organello footprinting pattern of the termination region and on in vitro transcription termination, strongly suggesting that ATP control of rRNA synthesis occurs at the initiation level. Several methylation interference sites were found upstream of the whole H-strand transcription unit, pointing to possible protein-DNA interactions related to the activity of this unit. In vivo footprinting of the rDNA transcription termination region of human mtDNA has revealed a very strong protection pattern, indicating a high degree of occupancy of the termination site by mitochondrial transcription termination factor (~80%).

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Additional Information:©1997 by The American Society for Biochemistry and Molecular Biology, Inc. (Received for publication, December 23, 1996, and in revised form, April 14, 1997) We are very grateful to Giovanni Lesa for collaboration in the early stages of the investigations and to Carlo Ausenda for help in the experiments involving ethidium bromide treatment of HeLa cells. We also thank A. Drew and L. Tefo for expert technical assistance. This work was supported by National Institutes of Health Grant GM-11726 (to G. A.) and scholarships from the Spanish Ministry of Education (to V. M. and P. F.-S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Issue or Number:30
Record Number:CaltechAUTHORS:MICjbc97
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:3394
Deposited By: Archive Administrator
Deposited On:05 Jun 2006
Last Modified:02 Oct 2019 23:02

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