Kinetic characteristics of a pistol ribozyme. (A) Sequence and predicted secondary structure of the bimolecular construct derived from L. sphaericus. Altered nucleotides in mutant (M1, M3, M5) and compensatory mutant (M2, M4) constructs are denoted by boxes. The arrowhead designates the ribozyme-mediated cleavage site. Red nucleotides are highly conserved in the consensus model. (B) Self-cleavage activity of WT and mutant constructs with 32P-labeled substrate RNA. (NR) No reaction for the WT bimolecular construct. All other reactions were incubated under standard reaction conditions for 10 sec. (C) A representative time course for the bimolecular ribozyme construct under the conditions indicated as used for determining kobs values. (D) Effect of Mg2+ concentration on the rate of pistol ribozyme cleavage. (E) Effect of pH on the rate of pistol ribozyme cleavage.
