Purification of a functional enzymatic editing complex from Trypanosoma brucei mitochondria.
Kinetoplastid mitochondrial RNA editing, the insertion and deletion of U residues, is catalyzed by sequential cleavage, U addition or removal, and ligation reactions and is directed by complementary guide RNAs. We have purified a approximately 20S enzymatic complex from Trypanosoma brucei mitochondria that catalyzes a complete editing reaction in vitro. This complex possesses all four activities predicted to catalyze RNA editing: gRNA-directed endonuclease, terminal uridylyl transferase, 3' U-specific exonuclease, and RNA ligase. However, it does not contain other putative editing complex components: gRNA-independent endonuclease, RNA helicase, endogenous gRNAs or pre-mRNAs, or a 25 kDa gRNA-binding protein. The complex is composed of eight major polypeptides, three of which represent RNA ligase. These findings identify polypeptides representing catalytic editing factors, reveal the nature of this approximately 20S editing complex, and suggest a new model of editosome assembly.
Rusché
L N
LN
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Cruz-Reyes
J
J
Piller
K J
KJ
Sollner-Webb
B
B
D000818:Animals; D002458:Cell Fractionation; D002482:Cellulose; D004247:DNA; D004722:Endoribonucleases / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D017393:RNA Editing; D011130:RNA Ligase (ATP) / Q000302:isolation & purification / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D016053:RNA, Protozoan / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics
D009097:Multienzyme Complexes; D016053:RNA, Protozoan; D002482:Cellulose; D004247:DNA; D012316:RNA Nucleotidyltransferases; C031687:UTP-RNA uridylyltransferase; D004722:Endoribonucleases; D005092:Exonucleases; D011130:RNA Ligase (ATP)