FIGURE 2.

Kinetics of cleavage of TGR1 and the C75 variants. (A) Cleavage of TGR1 and C75 mutants in the absence of exogenous base in 10 mM MgCl2, 50 mM Tris-HCl (pH 7.5) at 37°C. The curves are fit to an exponential with rate constants of 19 min⁻¹ for the wild-type ribozyme (open circles) and 3.2 × 10⁻3 min⁻¹ for the C75A variant (closed circles). For the C75U and C75Δ variants, nonspecific degradation interfered with quantifying specific cleavage; limits for the rate constants were estimated at about 5 × 10⁻⁶ and 5 × 10⁻⁵ min⁻¹, respectively, from the linear fit. (B) Cleavage of the C75Δ variant rescued with exogenous cytosine at the pH given in the figure. Rescue reactions contained 25 mM MgCl₂, 37 mM cytosine, and 40 mM AMT/MTA buffers (see Materials and Methods).