Role of Nd3+ and Pb2+ on the RNA cleavage reaction by a small ribozyme.

Abstract

Leadzyme is a ribozyme that requires Pb2+. We have previously shown that the addition of Nd3+ in the presence of Pb2+ increased significantly the yield of the RNA cleavage reaction by a leadzyme, although other rare earth ions or divalent ions except Pb2+ did not promote the reaction [Sugimoto, N., & Ohmichi, T. (1996) FEBS Lett. 393, 97-100]. To investigate the combined effects of Nd3+ and Pb2+ on the binding and cleavage steps of a leadzyme, CUGGGAGUCC, with a substrate, GGACCGAGCCAG, kinetics for the leadzyme reaction have been measured at various concentration ratios of Nd3+ and Pb2+. At low concentration ratios of Nd3+ under a constant total concentration of metal ions, Nd3+ increased the stability of the complex between the leadzyme and the substrate. In contrast, at high concentration ratios of Nd3+, the addition of Nd3+ decreased the stability of the complex. The rate constant of the cleavage step was maximized when the ratio of Nd3+ to Pb2+ was 1:1. These results suggest that the complex between the leadzyme and the substrate has binding sites for Nd3+ ion that influence complex stability and catalyze directly the cleavage reaction. On the basis of the results, we propose a two-metal-ion mechanism in which Pb2+ and Nd3+ play the roles of base and acid catalyst, respectively.