A base triple in the Tetrahymena group I core affects the reaction equilibrium via a threshold effect

Abstract

Previous work on group I introns has suggested that a central base triple might be more important for the first rather than the second step of self-splicing, leading to a model in which the base triple undergoes a conformational change during self-splicing. Here, we use the well-characterized L-21 ScaI ribozyme derived from the Tetrahymena group I intron to probe the effects of base-triple disruption on individual reaction steps. Consistent with previous results, reaction of a ternary complex mimicking the first chemical step in self-splicing is slowed by mutations in this base triple, whereas reaction of a ternary complex mimicking the second step of self-splicing is not. Paradoxically, mechanistic dissection of the base-triple disruption mutants indicates that active site binding is weakened uniformly for the 5'-splice site and the 5'-exon analog, mimics for the species bound in the first and second step of self-splicing. Nevertheless, the 5'-exon analog remains bound at the active site, whereas the 5'-splice site analog does not. This differential effect arises despite the uniform destabilization, because the wild-type ribozyme binds the 5'-exon analog more strongly in the active site than in the 5'-splice site analog. Thus, binding into the active site constitutes an additional barrier to reaction of the 5'-splice site analog, but not the 5'-exon analog, resulting in a reduced reaction rate constant for the first step analog, but not the second step analog. This threshold model explains the self-splicing observations without the need to invoke a conformational change involving the base triple, and underscores the importance of quantitative dissection for the interpretation of effects from mutations.

FIGURE 1.

Secondary structure of the L-21 ScaI group I ribozyme from Tetrahymena thermophila. This schematic representation shows the secondary structure in the topology of the tertiary structure (Golden et al. 1998). The (G₁₁₁·C₂₀₉)·U₃₀₅ base triple is highlighted in bold, as are P1 and P9.0 formed in trans with oligonucleotide substrate S and UCG, respectively. The RNA core that is conserved between all subclasses of group I introns is shown in the gray-shaded area (Michel and Westhof 1990).

FIGURE 2.

Thermodynamic framework for the group I ribozyme reaction (e.g., Herschlag and Cech 1990; Bevilacqua et al. 1992; McConnell et al. 1993). (E^s)_o and (E^s)_c [or (E^P)_o and (E^P)_c] refer to the open and closed conformations of the P1 duplex, in which tertiary interactions between the ribozyme core are absent and present, respectively.

FIGURE 3.

Quantitative free-energy profile for the group I reaction with wild-type and (C₁₁₁·G₂₀₉) ribozyme. The free-energy profiles were calculated at 30°C (pH 7.2) and for a standard state of 1 mM G and GA. In the case of the reaction with wild-type ribozyme (continuous line), the reaction described by (k_cat/K_m)^G is from (E·S)_c + G and the transition state for the chemical step (‡). For the (C₁₁₁·G₂₀₉) mutant (broken line), the reaction described by (k_cat/K_m)^G is from (E·S)_o + G, and the transition state of the chemical step (‡), and thus includes the additional barrier for docking. The free-energy profile was calculated as outlined in the Materials and Methods using the values listed in Table 6 ▶.

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