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.

Keywords

• RNA structure
• catalysis
• threshold effects
• conformational changes
• base triple
• group I intron
• S, 5′-splice site analog without specifying the identity of the 2′-substituents, which can be a hydrogen atom, a hydroxyl group, or a methoxy group (see Chart 1 for the specific oligonucleotides used)
• P, 5′-exon analog (see Chart 1)
• *S, radioactively labeled S
• *P, radioactively labeled P
• G, guanosine
• E, L-21 ScaI ribozyme (mutant or wild type)
• HPLC, high pressure liquid chromatography
• HEPES, 4-(2-hydroxy-ethyl)-piperazine-1-ethane-sulfonic acid
• MOPS, 3-(N-morpholino)-propane-sulfonic acid
• EDTA, ethylenediaminetetraacetic acid, Tris, Tris[hydroxymethyl]aminomethane
• IGS, internal guide sequence