Distinct reaction pathway promoted by non-divalent-metal cations in a tertiary stabilized hammerhead ribozyme

Abstract

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 À1 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.

The Different Role of High-Affinity and Low-Affinity Metal Ions in Cleavage by a Tertiary Stabilized Cis Hammerhead Ribozyme from Tobacco Ringspot Virus

“…This suggests that these high-affi nity Mg 2+ /Mn 2+ ions are structurally necessary for the correct folding but are not suffi cient to establish full catalytic activity. This is in agreement with the statement that additional weakly bound divalent metal ions are needed for catalysis (Hammann and Lilley, 2002;Canny et al, 2004;Kim et al, 2005;Roychowdhury-Saha and Burke, 2007). Similar conclusions were derived from FRET experiments demonstrating that low concentrations of metal(II) ions are suffi cient for the one-step folding but not enough for catalysis (Penedo et al, 2004).…”

Dissecting metal ion–dependent folding and catalysis of a single DNAzyme

“…The two-fold reduction in the reaction rate compared to that of Pb 2+ only may arise due to competitive binding of Mg 2+ to a Pb 2+ binding site or due to the Mg 2+ -induced distortion of the conformations most active for Pb 2+ -dependent reaction. These results imply that there are multiple reaction pathways depending on the metal cofactors, similar to the proposals made for the hammerhead ribozyme 35 . The 8-17 DNAzyme in the presence of Zn 2+ or Mg 2+ ions behave similarly to many proteins and ribozymes in that it requires a metal-dependent global folding step before it can be functional.…”

Role of Mg2+ in Hammerhead Ribozyme Catalysis from Molecular Simulation

“…73 The proposed model derived from the simulations is consistent with the Mg 2+ having an active role in the chemical steps of catalysis and involving direct coordination to the A9 and scissile phosphates.…”