title: Novel mechanism of RNA repair by RtcB via sequential 2',3'-cyclic phosphodiesterase and 3'-Phosphate/5'-hydroxyl ligation reactions.
abstract: RtcB enzymes are a newly discovered family of RNA ligases, implicated in tRNA splicing and other RNA repair reactions, that seal broken RNAs with 2',3'-cyclic phosphate and 5'-OH ends. Parsimony and energetics would suggest a one-step mechanism for RtcB sealing via attack by the O5' nucleophile on the cyclic phosphate, with expulsion of the ribose O2' and generation of a 3',5'-phosphodiester at the splice junction. Yet we find that RtcB violates Occam's razor, insofar as (i) it is adept at ligating 3'-monophosphate and 5'-OH ends; (ii) it has an intrinsic 2',3'-cyclic phosphodiesterase activity. The 2',3'-cyclic phosphodiesterase and ligase reactions both require manganese and are abolished by mutation of the RtcB active site. Thus, RtcB executes a unique two-step pathway of strand joining whereby the 2',3'-cyclic phosphodiester end is hydrolyzed to a 3'-monophosphate, which is then linked to the 5'-OH end to form the splice junction. The energy for the 3'-phosphate ligase activity is provided by GTP, which reacts with RtcB in the presence of manganese to form a covalent RtcB-guanylate adduct. This adduct is sensitive to acid and hydroxylamine but resistant to alkali, consistent with a phosphoramidate bond.
authors: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.
authors: Naoko
authors: N
authors: Tanaka
authors: Anupam K
authors: AK
authors: Chakravarty
authors: Bill
authors: B
authors: Maughan
authors: Stewart
authors: S
authors: Shuman
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009708:Nucleotidases / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D012326:RNA Splicing / Q000235:genetics / Q000502:physiology
chemical_list: D029968:Escherichia coli Proteins; D008345:Manganese; D012313:RNA; D006160:Guanosine Triphosphate; D009708:Nucleotidases; C040470:3'-nucleotidase; D000604:Amino Acyl-tRNA Synthetases; C561935:RtcB protein, E coli