A base triple in the Tetrahymena group I core affects the reaction equilibrium via a threshold effect.
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.
Karbstein
Katrin
K
Stanford University, School of Medicine, Beckman Center B400, Department of Biochemistry, 279 Campus Drive, Stanford, CA 94305, USA.
Tang
Kuo-Hsiang
KH
Herschlag
Daniel
D
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D005091:Exons; D007438:Introns; D007700:Kinetics; D008954:Models, Biological; D009154:Mutation; D009690:Nucleic Acid Conformation; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016053:RNA, Protozoan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013768:Tetrahymena / Q000235:genetics* / Q000378:metabolism*; D013816:Thermodynamics
D016337:RNA, Catalytic; D016053:RNA, Protozoan