RNA editing provides a powerful means of altering gene expression in many systems, including humans, and results in RNA products which differ in highly specific ways from their original templates. Most RNAs present in the mitochondria of Physarum contain non-encoded residues, and insertion of each of the four nucleotides has been observed in this system. The long term goal of this project is to determine the mechanism by which this precise insertion of nucleotides is accomplished. Experiments with isolated mitochondria have demonstrated that editing in Physarum is closely associated with RNA synthesis. Transcription complexes isolated from Physarum mitochondria retain the ability to synthesize edited RNA, permitting the use of these complexes in mechanistic studies. The relationship between transcription and editing will be examined further by manipulating polymerase elongation rates and availability of nucleotide substrates, and through the use of chain-terminating nucleotide analogs. Both sets of experiments should yield information as to the point of nucleotide addition during synthesis of nascent RNAs. Isolated transcription complexes will also be subjected to strong washing conditions to determine whether there are dissociable trans-acting editing factors involved in the insertion of non-encoded nucleotides. Finally, further steps will be taken toward the establishment of a soluble in vitro transcription-editing system. Mitochondrial promoters will be identified and any necessary factor(s) required for their utilization will be isolated. The ultimate aim of these experiments is a molecular description of insertional editing in Physarum, including the identification of cis-acting sequences, trans-acting factors, and the chemical steps involved in this extraordinary process.
