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J. Biol. Chem., Vol. 280, Issue 27, 25499-25505, July 8, 2005

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The Human Mitochondrial Transcription Termination Factor (mTERF) Is Fully Active in Vitro in the Non-phosphorylated Form^*

Jordi Asin-Cayuela, Thomas Schwend, Géraldine Farge, and Claes M. Gustafsson

From the Department of Medical Nutrition, Karolinska Institutet, Novum, Huddinge Hospital, S-141 86 Huddinge, Sweden

The human mitochondrial transcription termination factor (mTERF) is a 39-kDa protein that terminates transcription at the 3'-end of the 16 S rRNA gene and thereby controls expression of the ribosomal transcription unit of mitochondrial DNA. The transcription termination activity of human mTERF has been notoriously difficult to study in vitro, and it has been suggested that the activity of the protein is regulated by posttranslational modifications or by protein polymerization. We here characterize the activity of recombinant human mTERF expressed in insect cells. We observed that mTERF efficiently promotes sequence-specific termination in a completely recombinant and highly purified in vitro system for mitochondrial transcription. The termination activity has a distinct polarity, and we observed complete transcription termination when the mTERF-binding site is oriented in a forward position relative the heavy strand promoter but only partial transcription termination when the binding site is in the reverse position. We analyzed the biochemical characteristics of the active mTERF protein and found that it is a stable monomer at physiological salt concentration. Structural analysis, including phosphostaining, two-dimensional electrophoresis, and electrospray mass spectrometry, detected no evidence of phosphorylation. We conclude that the monomeric human mTERF is fully active in its non-phosphorylated form and that the protein does not require additional cellular factors to terminate mitochondrial transcription in vitro.

Received for publication, February 1, 2005 , and in revised form, April 27, 2005.

^* This work was supported by grants from the Swedish Research Council, the Swedish Cancer Society, the Swedish Foundation for Strategic Research, the Swedish Agency for Innovation Systems (VINNOVA), and the European Union Sixth Framework Program (EUMITOCOMBAT) (to C. M. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a Marie Curie Intra-European Fellowship from the European Union Sixth Framework Program.

To whom correspondence should be addressed. Tel.: 46-8-5858-3974; Fax: 46-8-779-5383; E-mail: claes.gustafsson{at}mednut.ki.se.

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