In vitro RNA editing in pea mitochondria requires NTP or dNTP, suggesting involvement of an RNA helicase.
To analyze the biochemical parameters of RNA editing in plant mitochondria and to eventually characterize the enzymes involved we developed a novel in vitro system. The high sensitivity of the mismatch-specific thymine glycosylase is exploited to facilitate reliable quantitative evaluation of the in vitro RNA editing products. A pea mitochondrial lysate correctly processes a C to U editing site in the cognate atp9 template. Reaction conditions were determined for a number of parameters, which allow first conclusions on the proteins involved. The apparent tolerance against specific Zn2+ chelators argues against the involvement of a cytidine deaminase enzyme, the theoretically most straightforward catalysator of the deamination reaction. Participation of a transaminase was investigated by testing potential amino group receptors, but none of these increased the RNA editing reaction. Most notable is the requirement of the RNA editing activity for NTPs. Any NTP or dNTP can substitute for ATP to the optimal concentration of 15 mm. This observation suggests the participation of an RNA helicase in the predicted RNA editing protein complex of plant mitochondria.
Takenaka
Mizuki
M
Molekulare Botanik, Universität Ulm, 89069 Ulm, Germany. mizuki.takenaka@biologie.uni-ulm.de
Brennicke
Axel
A
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000494:pharmacology; D020137:Base Pair Mismatch; D002614:Chelating Agents / Q000494:pharmacology; D003564:Cytidine Deaminase / Q000737:chemistry; D004260:DNA Repair; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D066298:In Vitro Techniques; D007477:Ions / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D008957:Models, Genetic; D009711:Nucleotides / Q000737:chemistry*; D018532:Pisum sativum / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D012313:RNA / Q000737:chemistry; D017393:RNA Editing; D020365:RNA Helicases / Q000737:chemistry*; D013997:Time Factors; D015032:Zinc / Q000737:chemistry
D002614:Chelating Agents; D018076:DNA, Complementary; D007477:Ions; D009711:Nucleotides; D012313:RNA; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D003564:Cytidine Deaminase; D020365:RNA Helicases; D015032:Zinc