Novel mechanism of RNA repair by RtcB via sequential 2',3'-cyclic phosphodiesterase and 3'-Phosphate/5'-hydroxyl ligation reactions

J Biol Chem. 2011 Dec 16;286(50):43134-43. doi: 10.1074/jbc.M111.302133. Epub 2011 Oct 31.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acyl-tRNA Synthetases / genetics
  • Amino Acyl-tRNA Synthetases / metabolism*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Guanosine Triphosphate / metabolism
  • Manganese / metabolism
  • Mutagenesis, Site-Directed
  • Nucleotidases / genetics
  • Nucleotidases / metabolism*
  • RNA / metabolism*
  • RNA Splicing / genetics
  • RNA Splicing / physiology

Substances

  • Escherichia coli Proteins
  • Manganese
  • RNA
  • Guanosine Triphosphate
  • Nucleotidases
  • 3'-nucleotidase
  • Amino Acyl-tRNA Synthetases
  • RtcB protein, E coli