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

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 Mg2+ for activity and has a [Mg2+](1/2) of 70-100 mM. It has an unusual specificity for Mg2+; there is only marginal activity in Mn2+ and no detectable activity in Ca2+, Sr2+, Ba2+, Zn2+, Co2+, Cd2+, Pb2+, Co(NH3)(6)(3+), or spermine. All tested cations other than Mg2+, including Mn2+, inhibit the ribozyme. Hill analysis in the presence of inhibitory cations suggested that Ca2+ and Co(NH3)(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 Ca2+ or Co(NH3)(6)(3+), as observed by hydroxyl-radical mapping. As further support for a nativelike fold in Ca2+, ribozyme that has been prefolded in Ca2+ can carry out the self-ligation very quickly upon the addition of Mg2+. Ligation rates of the prefolded ribozyme were directly measured and proceed at 800 min(-1) at pH 9.0.