Quantitative studies of Mn(2+)-promoted specific and non-specific cleavages of a large RNA: Mn(2+)-GAAA ribozymes and the evolution of small ribozymes.
Manganese (Mn(2+)) 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 Mn(2+)-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 Mn(2+) concentration. Interestingly, at site III, Mg(2+) alone promoted weak, but the same specific cleavage as Mn(2+). When added with Mn(2+), Mg(2+) had a synergistic effect on cleavage at site III, but inhibited cleavage at the other sites. Mn(2+) cleavage at site III also exhibited lower values of K (Mn(2+) 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 Mn(2+)-GAAA ribozyme. The implications for evolution of small ribozymes are also discussed.
Kuo
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Section of Molecular Cell and Developmental Biology and Institute for Cellular and Molecular Biology, BIO 311 24th Street and Whitis Avenue, University of Texas at Austin, Austin, TX 78712, USA.
Herrin
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D001483:Base Sequence; D002384:Catalysis / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D019143:Evolution, Molecular; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology*; D009154:Mutation / Q000235:genetics; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature
D016337:RNA, Catalytic; D012338:RNA, Ribosomal, 23S; D008345:Manganese; D008274:Magnesium