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.
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