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 fo ur minor (II, IV, V and VI) sites, in addition to slow non-specific cleavag e, in a 659-nucleotide RNA containing the Cr.LSU group I intron. The specif ic cleavages occurred between G and AAA sequences and thus can be considere d Mn2+-GAAA ribozymes. We have estimated rates of specific and non-specific cleavages under different conditions, Comparisons of the rates of major-sp ecific and background cleavages gave a maximal specificity of approximately 900 for GAAA cleavage. Both specific and non-specific cleavages showed hyp erbolic kinetics and there was no evidence of cooperativity with Mn2+ conce ntration. Interestingly, at site III, Mg2+ alone promoted weak, but the sam e specific cleavage as Mn2+. When added with Mn2+, Mg2+ had a synergistic e ffect on cleavage at site III, but inhibited cleavage at the other sites, M n2+ cleavage at site III also exhibited lower values of K-1/2 (Mn2+ require ment), pH-dependency and activation energy than did cleavage at the other s ites. In contrast, the pH-dependency and activation energy for cleavage at site I was similar to non-specific cleavage. These results increase our und erstanding of the Mn2+-GAAA ribozyme, The implications for evolution of sma ll ribozymes are also discussed.