Trypanosoma brucei mitochondria contain RNA helicase activity.
Mitochondrial gene expression in kinetoplastid organisms such as Trypanosoma, Leishmania and Crithidia requires a posttranscriptional RNA processing event known as kRNA editing. During editing, uridine nucleotides get inserted and deleted into pre-mRNAs directed by small, metabolically stable RNAs, termed guide RNAs. Although the precise mechanism of the reaction is not understood, the accepted working model describes the formation of extended anti-parallel RNA helices between gRNA molecules with pre- and partially edited mRNAs as intermediates. These duplex structures must be separated to ensure the sequential action of multiple gRNAs in a 3' to 5' polarity on the mRNA molecule. In spite of this fact, no unwinding activity has heretofore been identified in kinetoplastid mitochondria. We report the characterisation of a RNA helicase activity within Trypanosoma brucei mitochondrial extracts. The activity unwinds 25- and 48 bp, tailed RNA duplex structures but fails to separate DNA strands. It can be destroyed by heat denaturation as well as by proteinase K treatment. The activity requires magnesium cations and acts in a NTP/dNTP dependent manner. Hydrolysis of a nucleoside triphosphate is required rather than mere NTP binding as deduced from a comparison of unwinding in the presence of ATP and AMP-PCP. RNA duplexes mimicking presumed kRNA editing intermediates are substrates of the unwinding activity and therefore, we address the possible involvement of a RNA helicase activity during kRNA editing.
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Laboratorium für Molekulare Biologie--Genzentrum, Universität München am Max-Planck-Institut für Biochemie, Martinsried, Germany.
Göringer
H U
HU
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D019286:Endopeptidase K; D006358:Hot Temperature; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011489:Protein Denaturation; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D017393:RNA Editing; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000494:pharmacology; D013379:Substrate Specificity; D013816:Thermodynamics; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000648:ultrastructure
D012313:RNA; D000255:Adenosine Triphosphate; D012316:RNA Nucleotidyltransferases; D012697:Serine Endopeptidases; D019286:Endopeptidase K; D020365:RNA Helicases; D008274:Magnesium