Published online 4 August 2004
Nucleic Acids Research, Vol. 32 No. 14 © Oxford University Press 2004; all rights reserved
Complex cis-elements determine an RNA editing site in pea mitochondria
Molekulare Botanik, Universität Ulm, 89069 Ulm, Germany
* To whom correspondence should be addressed. Tel: +49 731 502 2616; Fax: +49 731 502 2626; Email: mizuki.takenaka{at}biologie.uni-ulm.de
Received June 14, 2004; Revised and Accepted July 26, 2004
The cis-requirements for the first editing site in the atp9 mRNA from pea mitochondria were investigated in an in vitro RNA editing system. Template RNAs deleted 5' of –20 are edited correctly, but with decreased efficiency. Deletions between –20 and the edited nucleotide abolish editing activity. Substitution of the sequences 3' of the editing site has little effect, which suggests that the major determinants reside upstream. Stepwise mutated RNA sequences were used as templates or competitors that divide the cis-elements into several distinct regions. In the template RNAs, mutation of the sequence between –40 and –35 reduces the editing activity, while the region from –15 to –5 is essential for the editing reaction. In competition experiments the upstream region can be titrated, while the essential sequence near the editing site is largely resistant to excess competitor. This observation suggests that either one trans-factor attaches to these separate cis-regions with different affinities or two distinct trans-factors bind to these sequences, and one of which is present in limited amounts, wheras the other one is more abundant in the lysate.
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