Nucleic Acids Research, 2000, Vol. 28, No. 19 3695-3701
© 2000 Oxford University Press
Transcription and RNA editing in a soluble in vitro system from Physarum mitochondria
Center for RNA Molecular Biology, Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106, USA
The dissection of RNA editing mechanisms in Physarum mitochondria has been hindered by the absence of a soluble in vitro system. Based on our studies in isolated mitochondria, insertion of non-encoded nucleotides into Physarum mitochondrial RNAs is closely linked to transcription. Here we have fractionated mitochondrial lysates, enriching for run-on RNA synthesis, and find that editing activity co-fractionates with pre-formed transcription elongation complexes. The establishment of this soluble transcription-editing system allows access to the components of the editing machinery and permits manipulation of transcription and editing substrates. Thus, the availability of this system provides, for the first time, a means of investigating roles for cis-acting elements, trans-acting factors and nucleotide requirements for the insertion of non-encoded nucleotides into Physarum mitochondrial RNAs. This methodology should also be broadly applicable to the study of RNA processing and editing mechanisms in a wide range of mitochondrial systems.
* To whom correspondence should be addressed. Tel: +1 216 368 3930; Fax: +1 216 368 3055; Email: jmg13@po.cwru.edu
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