Transcription and RNA editing in a soluble in vitro system from Physarum mitochondria

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 Physaru m mitochondrial RNAs is closely linked to transcription. Here we have fract ionated mitochondrial lysates, enriching for run-on RNA synthesis, and find that editing activity cofractionates with pre-formed transcription elongat ion complexes, The establishment of this soluble transcription-editing syst em allows access to the components of the editing machinery and permits man ipulation of transcription and editing substrates, Thus, the availability o f this system provides, for the first time, a means of investigating roles for cis-acting elements, trans-acting factors and nucleotide requirements f or the insertion of non-encoded nucleotides into Physarum mitochondrial RNA s, This methodology should also be broadly applicable to the study of RNA p rocessing and editing mechanisms in a wide range of mitochondrial systems.