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J. Biol. Chem., Vol. 278, Issue 48, 47526-47533, November 28, 2003

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In Vitro RNA Editing in Pea Mitochondria Requires NTP or dNTP, Suggesting Involvement of an RNA Helicase^*

Mizuki Takenaka and Axel Brennicke

From the Molekulare Botanik, Universität Ulm, 89069 Ulm, Germany

To analyze the biochemical parameters of RNA editing in plant mitochondria and to eventually characterize the enzymes involved we developed a novel in vitro system. The high sensitivity of the mismatch-specific thymine glycosylase is exploited to facilitate reliable quantitative evaluation of the in vitro RNA editing products. A pea mitochondrial lysate correctly processes a C to U editing site in the cognate atp9 template. Reaction conditions were determined for a number of parameters, which allow first conclusions on the proteins involved. The apparent tolerance against specific Zn²⁺ chelators argues against the involvement of a cytidine deaminase enzyme, the theoretically most straightforward catalysator of the deamination reaction. Participation of a transaminase was investigated by testing potential amino group receptors, but none of these increased the RNA editing reaction. Most notable is the requirement of the RNA editing activity for NTPs. Any NTP or dNTP can substitute for ATP to the optimal concentration of 15 mM. This observation suggests the participation of an RNA helicase in the predicted RNA editing protein complex of plant mitochondria.

Received for publication, May 21, 2003 , and in revised form, September 2, 2003.

^* This work was supported by a grant from the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie. 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.

To whom correspondence should be addressed. Tel.: 49-731-502-2616; Fax: 49-731-502-2626; E-mail: mizuki.takenaka{at}biologie.uniulm.de.

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