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.
Highlights
RtcB is a new RNA ligase implicated in tRNA splicing and RNA repair
It was shown that the 2Ј,3Ј-cyclic phosphate of the cleaved pre-tRNA is incorporated at the splice junction in the mature tRNA. (By contrast, the junction phosphate in yeast tRNA splicing derives from the ␥-phosphate of the NTP substrate for the 5Ј kinase step.) Direct ligation is thought to be the main pathway of tRNA splicing in animals and archaea [13,14,15]
RtcB Seals Stem-loops with 3Ј-Monophosphate/5Ј-OH Breaks—We showed previously that RtcB was adept at ligating broken stem-loop structures with 2Ј,3Ј-cyclic phosphate and 5Ј-OH termini to restore a 3Ј,5Ј-phosphodiester at the repair junction [18]
Summary
RtcB is a new RNA ligase implicated in tRNA splicing and RNA repair. Results: RtcB has 2Ј,3Ј-cyclic phosphodiesterase and 3Ј-phosphate/5Ј-hydroxyl ligase activities. 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. (By contrast, the junction phosphate in yeast tRNA splicing derives from the ␥-phosphate of the NTP substrate for the 5Ј kinase step.) Direct ligation is thought to be the main pathway of tRNA splicing in animals and archaea [13,14,15] Earlier this year, three laboratories reported the identification of bacterial, archaeal, and mammalian RtcB proteins as novel RNA ligase enzymes capable of sealing 2Ј,3Ј-cyclic phosphate and 5Ј-OH ends in the absence of ATP (16 –18).
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