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 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.
Glasner
Margaret E
ME
Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA 02142, USA.
Bergman
Nicholas H
NH
Bartel
David P
DP
D001483:Base Sequence; D002118:Calcium / Q000737:chemistry; D002384:Catalysis; D002413:Cations, Divalent / Q000737:chemistry; D002414:Cations, Monovalent / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011188:Potassium / Q000737:chemistry; D011130:RNA Ligase (ATP) / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D016337:RNA, Catalytic / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
D002413:Cations, Divalent; D002414:Cations, Monovalent; D004791:Enzyme Inhibitors; D008670:Metals; D016337:RNA, Catalytic; D011130:RNA Ligase (ATP); D008274:Magnesium; D011188:Potassium; D002118:Calcium