Distinctive kinetics and substrate specificities of plant and fungal tRNA ligases

RNA. 2014 Apr;20(4):462-73. doi: 10.1261/rna.043752.113. Epub 2014 Feb 19.

Abstract

Plant and fungal tRNA ligases are trifunctional enzymes that repair RNA breaks with 2',3'-cyclic-PO4 and 5'-OH ends. They are composed of cyclic phosphodiesterase (CPDase) and polynucleotide kinase domains that heal the broken ends to generate the 3'-OH, 2'-PO4, and 5'-PO4 required for sealing by a ligase domain. Here, we use short HORNA>p substrates to determine, in a one-pot assay format under single-turnover conditions, the order and rates of the CPDase, kinase and ligase steps. The observed reaction sequence for the plant tRNA ligase AtRNL, independent of RNA length, is that the CPDase engages first, converting HORNA>p to HORNA2'p, which is then phosphorylated to pRNA2'p by the kinase. Whereas the rates of the AtRNL CPDase and kinase reactions are insensitive to RNA length, the rate of the ligase reaction is slowed by a factor of 16 in the transition from 10-mer RNA to 8-mer and further by eightfold in the transition from 8-mer RNA to 6-mer. We report that a single ribonucleoside-2',3'-cyclic-PO4 moiety enables AtRNL to efficiently splice an otherwise all-DNA strand. Our characterization of a fungal tRNA ligase (KlaTrl1) highlights important functional distinctions vis à vis the plant homolog. We find that (1) the KlaTrl1 kinase is 300-fold faster than the AtRNL kinase; and (2) the KlaTrl1 kinase is highly specific for GTP or dGTP as the phosphate donor. Our findings recommend tRNA ligase as a tool to map ribonucleotides embedded in DNA and as a target for antifungal drug discovery.

Keywords: 2′,3′ cyclic phosphodiesterase; RNA ligase; RNA repair; polynucleotide kinase.

Publication types

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

MeSH terms

  • Arabidopsis / enzymology*
  • Arabidopsis / genetics
  • Kinetics
  • Kluyveromyces / enzymology
  • Kluyveromyces / genetics
  • Phosphoric Diester Hydrolases / genetics
  • Phosphoric Diester Hydrolases / metabolism*
  • Polynucleotide 5'-Hydroxyl-Kinase / genetics
  • Polynucleotide 5'-Hydroxyl-Kinase / metabolism*
  • RNA / chemistry
  • RNA / genetics*
  • RNA Ligase (ATP) / genetics
  • RNA Ligase (ATP) / isolation & purification
  • RNA Ligase (ATP) / metabolism*
  • RNA Splicing / genetics
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics
  • Substrate Specificity

Substances

  • RNA
  • Polynucleotide 5'-Hydroxyl-Kinase
  • Phosphoric Diester Hydrolases
  • RNA Ligase (ATP)