Effects of cobalt hexammine on folding and self-cleavage of the Neurospora VS ribozyme

Abstract

We have investigated the effects of Co(NH 3) 6 3+, an analog of hexahydrated Mg 2+, on folding and catalysis of the Neurospora VS ribozyme. Most of the metal ion-induced changes detected by chemical modification structure probing in either metal ion are similar, but occur at approximately 33-fold lower concentrations of Co(NH 3) 6 3+ than Mg 2+. However, Co(NH 3) 6 3+ is not as effective at inducing two functionally important structural changes: stabilizing the pseudoknot interaction between loops I and V, and rearranging the secondary structure of helix Ib. Comparison of the folding of the precursor and the downstream cleavage product, which lacks helix Ia, shows that helix Ia inhibits stable pseudoknot formation and rearrangement of helix Ib. The VS ribozyme does not self-cleave with Co(NH 3) 6 3+ as the sole polyvalent cation; however, mixed-metal kinetic experiments show that Co(NH 3) 6 3+ does not inhibit Mg 2+-induced self-cleavage. In contrast, at sub-saturating concentrations of Mg 2+, Co(NH 3) 6 3+ increases the rate of Mg 2+-induced self-cleavage, indicating that Co(NH 3) 6 3+ contributes to the functionally relevant folding of the VS ribozyme.