Distinct reaction pathway promoted by non-divalent-metal cations in a tertiary stabilized hammerhead ribozyme.
Divalent ion sensitivity of hammerhead ribozymes is significantly reduced when the RNA structure includes appropriate tertiary stabilization. Therefore, we investigated the activity of the tertiary stabilized "RzB" hammerhead ribozyme in several nondivalent ions. Ribozyme RzB is active in spermidine and Na(+) alone, although the cleavage rates are reduced by more than 1,000-fold relative to the rates observed in Mg(2+) and in transition metal ions. The trivalent cobalt hexammine (CoHex) ion is often used as an exchange-inert analog of hydrated magnesium ion. Trans-cleavage rates exceeded 8 min(-1) in 20 mM CoHex, which promoted cleavage through outersphere interactions. The stimulation of catalysis afforded by the tertiary structural interactions within RzB does not require Mg(2+), unlike other extended hammerhead ribozymes. Site-specific interaction with at least one Mg(2+) ion is suggested by CoHex competition experiments. In the presence of a constant, low concentration of Mg(2+), low concentrations of CoHex decreased the rate by two to three orders of magnitude relative to the rate in Mg(2+) alone. Cleavage rates increased as CoHex concentrations were raised further, but the final fraction cleaved was lower than what was observed in CoHex or Mg(2+) alone. These observations suggest that Mg(2+) and CoHex compete for binding and that they cause misfolded structures when they are together. The results of this study support the existence of an alternate catalytic mechanism used by nondivalent ions (especially CoHex) that is distinct from the one promoted by divalent metal ions, and they imply that divalent metals influence catalysis through a specific nonstructural role.
Roychowdhury-Saha
Manami
M
Department of Chemistry, Indiana University, Bloomington, Indiana 47405, USA.
Burke
Donald H
DH
D001483:Base Sequence; D001667:Binding, Competitive; D002412:Cations / Q000378:metabolism; D003035:Cobalt / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008954:Models, Biological; D009690:Nucleic Acid Conformation; D020871:RNA Stability; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D013095:Spermidine / Q000378:metabolism
D002412:Cations; D016337:RNA, Catalytic; C427204:hammerhead ribozyme; C032300:hexaamminecobalt(II); D003035:Cobalt; D012964:Sodium; D008274:Magnesium; D013095:Spermidine