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 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.
Cheng
Y W
YW
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.
Gott
J M
JM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002457:Cell Extracts; D002474:Cell-Free System; D008928:Mitochondria / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000235:genetics / Q000378:metabolism; D010804:Physarum / Q000737:chemistry / Q000166:cytology / Q000201:enzymology / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D017393:RNA Editing / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D016053:RNA, Protozoan / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D012995:Solubility; D014158:Transcription, Genetic / Q000235:genetics
D002457:Cell Extracts; D009711:Nucleotides; D012333:RNA, Messenger; D016053:RNA, Protozoan; D000255:Adenosine Triphosphate; D012260:Ribonucleases