title: Mutational analysis of Trypanosoma brucei RNA editing ligase reveals regions critical for interaction with KREPA2.
abstract: The Trypanosoma brucei parasite causes the vector-borne disease African sleeping sickness. Mitochondrial mRNAs of T. brucei undergo posttranscriptional RNA editing to make mature, functional mRNAs. The final step of this process is catalyzed by the essential ligase, T. brucei RNA Editing Ligase 1 (TbREL1) and the closely related T. brucei RNA Editing Ligase 2 (TbREL2). While other ligases such as T7 DNA ligase have both a catalytic and an oligonucleotide/oligosaccharide-binding (OB)-fold domain, T. brucei RNA editing ligases contain only the catalytic domain. The OB-fold domain, which is required for interaction with the substrate RNA, is provided in trans by KREPA2 (for TbREL1) and KREPA1 (for TbREL2). KREPA2 enhancement of TbREL1 ligase activity is presumed to occur via an OB-fold-mediated increase in substrate specificity and catalysis. We characterized the interaction between TbREL1 and KREPA2 in vitro using full-length, truncated, and point-mutated ligases. As previously shown, our data indicate strong, specific stimulation of TbREL1 catalytic activity by KREPA2. We narrowed the region of contact to the final 59 C-terminal residues of TbREL1. Specifically, the TbREL1 C-terminal KWKE (441-444) sequence appear to coordinate the KREPA2-mediated enhancement of TbREL1 activities. N-terminal residues F206, T264 and Y275 are crucial for the overall activity of TbREL1, particularly for F206, a mutation of this residue also disrupts KREPA2 interaction. Thus, we have identified the critical TbREL1 regions and amino acids that mediate the KREPA2 interaction.
authors: Department of Biochemistry, McGill University, McIntyre Medical Building, 3655 Promenade Sir William Osler, Montreal, Quebec, H3G1Y6, Canada.
authors: Vaibhav
authors: V
authors: Mehta
authors: Department of Biochemistry, McGill University, McIntyre Medical Building, 3655 Promenade Sir William Osler, Montreal, Quebec, H3G1Y6, Canada.
authors: Rajashree
authors: R
authors: Sen
authors: Department of Biochemistry, McGill University, McIntyre Medical Building, 3655 Promenade Sir William Osler, Montreal, Quebec, H3G1Y6, Canada.
authors: Houtan
authors: H
authors: Moshiri
authors: Department of Biochemistry, McGill University, McIntyre Medical Building, 3655 Promenade Sir William Osler, Montreal, Quebec, H3G1Y6, Canada; Institute of Parasitology, McGill University, 21111 Lakeshore Road, Ste. Anne de Bellevue, Montreal, Quebec, H9X3V9, Canada; McGill Centre for Bioinformatics, McGill University, Duff Medical Building, 3775 University Street, Montreal, Quebec, H3A2B4, Canada.
authors: Reza
authors: R
authors: Salavati
mesh_terms: D000595:Amino Acid Sequence; D019729:Carbon-Oxygen Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D017354:Point Mutation; D011485:Protein Binding; D017393:RNA Editing; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics
chemical_list: D019729:Carbon-Oxygen Ligases