Metal ion requirements for structure and catalysis of an RNA ligase ribozyme

Biochemistry. 2002 Jun 25;41(25):8103-12. doi: 10.1021/bi012179b.


The class I ligase, a ribozyme previously isolated from random sequence, catalyzes a reaction similar to RNA polymerization, positioning its 5'-nucleotide via a Watson-Crick base pair, forming a 3',5'-phosphodiester bond between its 5'-nucleotide and the substrate, and releasing pyrophosphate. Like most ribozymes, it requires metal ions for structure and catalysis. Here, we report the ionic requirements of this self-ligating ribozyme. The ligase requires at least five Mg(2+) for activity and has a [Mg(2+)](1/2) of 70-100 mM. It has an unusual specificity for Mg(2+); there is only marginal activity in Mn(2+) and no detectable activity in Ca(2+), Sr(2+), Ba(2+), Zn(2+), Co(2+), Cd(2+), Pb(2+), Co(NH(3))(6)(3+), or spermine. All tested cations other than Mg(2+), including Mn(2+), inhibit the ribozyme. Hill analysis in the presence of inhibitory cations suggested that Ca(2+) and Co(NH(3))(6)(3+) inhibit by binding at least two sites, but they appear to productively fill a subset of the required sites. Inhibition is not the result of a significant structural change, since the ribozyme assumes a nativelike structure when folded in the presence of Ca(2+) or Co(NH(3))(6)(3+), as observed by hydroxyl-radical mapping. As further support for a nativelike fold in Ca(2+), ribozyme that has been prefolded in Ca(2+) can carry out the self-ligation very quickly upon the addition of Mg(2+). Ligation rates of the prefolded ribozyme were directly measured and proceed at 800 min(-1) at pH 9.0.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Base Sequence
  • Calcium / chemistry
  • Catalysis
  • Cations, Divalent / chemistry
  • Cations, Monovalent / chemistry
  • Enzyme Inhibitors / chemistry
  • Hydrogen-Ion Concentration
  • Kinetics
  • Magnesium / chemistry
  • Metals / chemistry*
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Potassium / chemistry
  • RNA Ligase (ATP) / antagonists & inhibitors
  • RNA Ligase (ATP) / chemistry*
  • RNA Ligase (ATP) / metabolism
  • RNA, Catalytic / antagonists & inhibitors
  • RNA, Catalytic / chemistry*
  • RNA, Catalytic / metabolism
  • Structure-Activity Relationship


  • Cations, Divalent
  • Cations, Monovalent
  • Enzyme Inhibitors
  • Metals
  • RNA, Catalytic
  • RNA Ligase (ATP)
  • Magnesium
  • Potassium
  • Calcium