Figure 2.

Measurement of the catalytic reaction. A, Experimental scheme. RNA is pre-folded (typically 10–50 mM Mg²⁺, 37–60 °C, 10–60 min) and then mixed with substrate. B, Measurement of substrate cleavage by the Tetrahymena ribozyme (Russell and Herschlag, 1999). The reaction included 500 nM oligonucleotide substrate (with trace 5′-radiolabeled substrate) and 200 nM pre-folded ribozyme and was performed at 37 °C, pH 7.0, 10 mM Mg²⁺, with 1 mM guanosine. The cleavage reaction gave a burst of 0.8 product/ribozyme, indicating that most of the ribozyme was catalytically active. The rate constant was ≥8 min^–1, faster than folding under these conditions. C, Simulated data showing biphasic kinetics from an internal equilibrium (K_Int) between substrate cleavage and ligation. The approach to equilibrium occurs rapidly (lower dashed line at 0.6), and then dissociation of one of the products drives the reaction forward to a [product]/[ribozyme] value of 1 (upper dashed line, amplitude of 0.4 for the slow phase). From eq. 3 and 4, K_Int = 1.5 (0.6/0.4) and the correction factor C_F = 0.6. Panel B reprinted from Russell and Herschlag (1999) with permission from Elsevier.