TABLE 2.

Effect of disruption of the base triple on binding of 5′-splice site and 5′-exon analogs (S and P)

Ribozyme	KSd(nM)	kc(min−1)	KdP (nM)	K−c (min−1)
Wild type	0.18 ± 0.02	0.036 ± 0.06	8.8 ·10−3± 2 3 ·10−3	0.22 ± 0.03
C305	3.4 ± 0.2	0.013 ± 0.05	0.19 ± 0.03	0.14 ± 0.01
(C111 ·G209)	2.8 ± 0.2	0.0070 ± 0.003	0.94 ± 0.06	0.25 ± 0.01
(C111 · G209) ·C305	0.51 ± 0.01	0.019 ± 0.02	8.4 ·10−3± 9 3 ·10−4	0.18 ± 0.02

All experiments were performed at 30°C and pH 7.2 using −1d,rSA and −1d,rP. Dissociation constant for S and P were calculated from the dissociation rate constants determined in gel-shift experiments and the association rate constant of 10⁸ M⁻¹ min⁻¹ (Herschlag and Cech, 1990; Knitt and Herschlag 1996; Karbstein et al 2002). k_c values were obtained from the plateau of reaction rate constant dependences on G concentration and represent the rate constant at saturating G and S. k_−c values were obtained from the plateau of the rate constant of the reverse reaction at high AUCGA concentration and represent the rate constant at saturating AUCGA and P. The faster rate constants for the reverse reaction relative to the forward reaction can be explained by the use of AUCGA. Control experiments show that AUCG reacts approximately fivefold faster than G (data not shown).