TbMP42 is a structure-sensitive ribonuclease that likely follows a metal ion catalysis mechanism.
RNA editing in African trypanosomes is characterized by a uridylate-specific insertion and/or deletion reaction that generates functional mitochondrial transcripts. The process is catalyzed by a multi-enzyme complex, the editosome, which consists of approximately 20 proteins. While for some of the polypeptides a contribution to the editing reaction can be deduced from their domain structure, the involvement of other proteins remains elusive. TbMP42, is a component of the editosome that is characterized by two C(2)H(2)-type zinc-finger domains and a putative oligosaccharide/oligonucleotide-binding fold. Recombinant TbMP42 has been shown to possess endo/exoribonuclease activity in vitro; however, the protein lacks canonical nuclease motifs. Using a set of synthetic gRNA/pre-mRNA substrate RNAs, we demonstrate that TbMP42 acts as a topology-dependent ribonuclease that is sensitive to base stacking. We further show that the chelation of Zn(2+) cations is inhibitory to the enzyme activity and that the chemical modification of amino acids known to coordinate Zn(2+) inactivates rTbMP42. Together, the data are suggestive of a Zn(2+)-dependent metal ion catalysis mechanism for the ribonucleolytic activity of rTbMP42.
Niemann
Moritz
M
Genetics, Darmstadt University of Technology, Schnittspahnstrasse 10, 64287 Darmstadt, Germany.
Brecht
Michael
M
Schlüter
Elke
E
Weitzel
Kerstin
K
Zacharias
Martin
M
Göringer
H Ulrich
HU
D000596:Amino Acids / Q000737:chemistry; D002384:Catalysis; D009690:Nucleic Acid Conformation; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism; D017393:RNA Editing; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism; D017394:RNA, Guide, Kinetoplastida / Q000737:chemistry; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry* / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism; D015032:Zinc / Q000737:chemistry*
D000596:Amino Acids; D015800:Protozoan Proteins; D012322:RNA Precursors; D012333:RNA, Messenger; D012261:Ribonucleoproteins; C437999:TbMP42 protein, Trypanosoma brucei; D012260:Ribonucleases; D015032:Zinc; D017394:RNA, Guide, Kinetoplastida