Time course of cleavage reaction and kinetic analysis for HR-HTR2C and HR-HTR2C-edit against unedited- and edited-HTR2C RNA fragments under single-turnover conditions. (A) Denaturing PAGE (15%) showing the time course of cleavage of HR-HTR2C-edit against HTR2C-ade (top) and HTR2C-ino (bottom). Positions of the resulting 5′-cleavage products (P) are indicated by arrowheads. (B) Diagrams representing the cleavage ratio as a function of reaction time. Each data for the set of ribozyme and substrate (ribozyme/substrate) are represented as squares (HR-HTR2C/HTR2C-ade), inverted triangles (HR-HTR2C/HTR2C-ino), triangles (HR-HTR2C-edit/HTR2C-ade), and circles (HR-HTR2C-edit/HTR2C-ino). The cleavage ratio at different time points was determined using quantitative scanning of corresponding gel bands and calculated using the expression P/(S+P). All data were obtained from two independent experiments. Fitting to a single-exponential equation generated progress curves for the cleavage reactions of HR-HTR2C/HTR2C-ino and HR-HTR2C-edit/HTR2C-ade, and the data obtained for HR-HTR2C/HTR2C-ade and HR-HTR2C-edit/HTR2C-ino were fitted to a double-exponential equation. Kinetic parameters obtained in this experiment are summarized in Table 1.
