Evidence for Processivity and Two-step Binding of the RNA Substrate from Studies of J1/2 Mutants of the Tetrahymena Ribozymet

J1J2 of the Tetrahymena ribozyme, a sequence of three A residues, connects the RNA-binding site to the catakflic core. Addition or deletion of bases from J1/2 improves turnover and substrate specificity in the site-specific endonuclease reaction catalyzed by this ribozyme: G2CCCUCUA5 (S) + G + GzCCCUCU (P) + GAS. These paradoxical enhancements are caused by decreased affinity of the ribozyme for S and P [Young, B., Herschlag, D., & Cech, T. R. (1991) Cell 67, 10071. An additional property of these mutant ribozymes, decreased fidelity of RNA cleavage, is now analyzed. (Fidelity is the ability to cleave at the correct phosphodiester bond within a particular RNA substrate.) Introduction of deoxy residues to give “chimeric” ribo/deoxyribooligonucleotides changes the positions of incorrect cleavage. Previous work indicated that S is bound to the ribozyme by both base pairing and tertiary interactions involving 2’-hydroxyl groups of S. The data herein strongly suggest that the P1 duplex, which consists of S base-paired with the 5‘ exon binding site of the ribozyme, can dock into tertiary interactions in different registers; different 2’-hydroxyl groups of S plug into tertiary contacts with the ribozyme in the different registers. It is concluded that the mutations decrease fidelity by increasing the probability of docking out of register relative to docking in the normal register, thereby giving cleavage at different positions along S. These data also show that the contribution of J1/2 to the tertiary interactions is indirect, not direct. Thus, a structural role of the nonconserved J1/2 is indicated: this sequence positions S to optimize tertiary binding interactions and to ensure cleavage at the phosphodiester bond corresponding to the 5’ splice site. Substitution of sulfur for the nonbridging pro-Rp oxygen atom at the normal cleavage site has no effect on (kCat/K,,Js but decreases the fraction of cleavage at the normal site in reactions catalyzed by the -3A mutant ribozyme, which has all three A residues of J1/2 removed, Thus, the ribozyme chooses where to cleave S after rate-limiting binding of S, indicating that docking can change after binding and suggesting that the ribozyme could act processively. Indeed, it is shown that the +2A ribozyme cleaves at one position along an RNA substrate and then, before releasing that RNA product, cleaves it again. The ability of the substrate helix to move from one binding register to another without dissociation leads to a two-step model for RNA binding to the ribozyme: an “open” complex, in which S is held only by base-pairing interactions, precedes a “closed” complex, in which the base-paired duplex is docked into tertiary interactions. The change from the open to the closed complex can be described as an induced-fit conformational change, one which is able to contribute to specificity. %e intron from Tetmhyrnena pre-rRNA excises itself in the absence of proteins or self-splices, as do several related group I introns (Kruger et al., 1982; Cech, 1990). Circularization of the excised intron showed that catalytic activity is contained within the intron (Zaug et al., 1983), and conversion of this intramolecular self-splicing reaction into an intermolecular reaction catalyzed by a shortened form of the intron, or “ribozyme”, has facilitated detailed investigations (Zaug & Cech, 1986; Zaug et al., 1988; McSwiggen & Cech, 1989; Herschlag & Cech, 1990a-c; Pyle et al., 1990; Herschlag et al., 199 1). The site-specific endonuclease reaction catalyzed by the ribozyme is analogous to the first step of splicing, with the 5‘ splice site provided in trans (Figure 1). J1/2 connects the 5‘ exon binding site of the ribozyme to the rest of the ribozyme (Figure 2). Even though J1/2 is not conserved in length or sequence among group I introns, mutations in this region have dramatic effects. These mutations (described in the right panel of Figure 2) decrease the binding