Quantitative studies of Mn²⁺-promoted specific and non-specific cleavages of a large RNA: Mn²⁺-GAAA ribozymes and the evolution of small ribozymes

Manganese (Mn²⁺) promotes specific cleavage at two major (I and III) and four minor (II, IV, V and VI) sites, in addition to slow non-specific cleavage, in a 659-nucleotide RNA containing the Cr.LSU group I intron. The specific cleavages occurred between G and AAA sequences and thus can be considered Mn²⁺-GAAA ribozymes. We have estimated rates of specific and non-specific cleavages under different conditions. Comparisons of the rates of major-specific and background cleavages gave a maximal specificity of approximately 900 for GAAA cleavage. Both specific and non-specific cleavages showed hyperbolic kinetics and there was no evidence of cooperativity with Mn²⁺ concentration. Interestingly, at site III, Mg²⁺ alone promoted weak, but the same specific cleavage as Mn²⁺. When added with Mn²⁺, Mg²⁺ had a synergistic effect on cleavage at site III, but inhibited cleavage at the other sites. Mn²⁺ cleavage at site III also exhibited lower values of K( 1/2 ) (Mn²⁺ requirement), pH-dependency and activation energy than did cleavage at the other sites. In contrast, the pH-dependency and activation energy for cleavage at site I was similar to non-specific cleavage. These results increase our understanding of the Mn²⁺-GAAA ribozyme. The implications for evolution of small ribozymes are also discussed.