Properties of an In Vitro Selected Pb²⁺ Cleavage Motif

The addition of Pb²⁺ to a small RNA molecule consisting of an asymmetric internal loop of six nucleotides results in site-specific cleavage followed by hydrolysis of the 2′,3′-cyclic phosphate intermediate [Pan, T., & Uhlenbeck, O. C. (1992) Nature 358, 560–563]. Here we show that the reaction is highly specific for Pb²⁺ and the cleavage rate increases exponentially with pH from 5.5 to 7.0, both in the presence and in the absence of Mg²⁺. This suggests that the reaction mechanism involves Pb²⁺ hydroxide acting as a base. Several sequence variants of the RNA are found to be equally active in both steps of the reaction, suggesting that they fold into a similar structure.