Trypanosoma brucei 20 S editosomes have one RNA substrate-binding site and execute RNA unwinding activity.
Editing of mitochondrial pre-mRNAs in African trypanosomes generates full-length transcripts by the site-specific insertion and deletion of uridylate nucleotides. The reaction is catalyzed by a 0.8 MDa multienzyme complex, the editosome. Although the binding of substrate pre-edited mRNAs and cognate guide RNAs (gRNAs) represents the first step in the reaction cycle, the biochemical and biophysical details of the editosome/RNA interaction are not understood. Here we show that editosomes bind full-length substrate mRNAs with nanomolar affinity in a nonselective fashion. The complexes do not discriminate-neither kinetically nor thermodynamically-between different mitochondrial pre-mRNAs or between edited and unedited versions of the same transcript. They also bind gRNAs and gRNA/pre-mRNA hybrid RNAs with similar affinities and association rate constants. Gold labeling of editosome-bound RNA in combination with transmission electron microscopy identified a single RNA-binding site per editosome. However, atomic force microscopy of individual pre-mRNA-editosome complexes revealed that multiple editosomes can interact with one pre-mRNA. Lastly, we demonstrate a so far unknown activity of the editing machinery: editosome-bound RNA becomes unfolded by a chaperone-type RNA unwinding activity.
Department of Molecular Genetics, Darmstadt University of Technology, 64287 Darmstadt, Germany.
Cordula
C
Böhm
Venkata Subbaraju
VS
Katari
Michael
M
Brecht
H Ulrich
HU
Göringer
D001665:Binding Sites; D046911:Macromolecular Substances / Q000737:chemistry / Q000648:ultrastructure; D018625:Microscopy, Atomic Force; D046529:Microscopy, Electron, Transmission; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D015800:Protozoan Proteins / Q000737:chemistry* / Q000648:ultrastructure; D012323:RNA Processing, Post-Transcriptional; D017394:RNA, Guide, Kinetoplastida / Q000737:chemistry / Q000648:ultrastructure; D012333:RNA, Messenger / Q000737:chemistry* / Q000648:ultrastructure; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000648:ultrastructure; D020349:Surface Plasmon Resonance; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D046911:Macromolecular Substances; D015800:Protozoan Proteins; D012333:RNA, Messenger; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan; D016601:RNA-Binding Proteins; C029305:mitochondrial messenger RNA; D017394:RNA, Guide, Kinetoplastida