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.