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The active site of group I self-splicing introns occurs at the interface of two proposed structural domains. In the Tetrahymena intron, half of the catalytic core resides within the independently-folding P4-P6 domain while the other half belongs to a putative domain that includes helices P3, P7, P8, and P9 (P3-P9). To determine whether the P3-P9 region of the intron can also fold independently, we used Fe(II)-EDTA and dimethyl sulfate to probe the solvent accessibility of separate fragments of the Tetrahymena intron. These RNAs self-assemble into an active complex in trans, enabling analysis of their structural features both alone and within the complex. Our results show that while the P3-P9 region of the intron retains its secondary structure, most of the tertiary interactions within this region do not form stably in the absence of the P4-P6 domain. This indicates that the P4-P6 domain induces folding in the P3-P9 region, organizing the catalytic cleft between them. Thus the P4-P6 domain provides a scaffold for the folding of the Tetrahymena intron core.

The catalytic core of Group I self-splicing introns has been proposed to consist of two structural domains, P4-P6 and P3-P9. Each contains helical segments and conserved unpaired nucleotides, and the isolated P4-P6 domain has been shown to have substantial native tertiary structure. The proposed tertiary structure domains of the Tetrahymena intron were synthesized separately and shown to self-assemble into a catalytically active complex. Surprisingly, the concentration dependence of these reactions revealed that the domains interact with nanomolar apparent dissociation constants, even though there is no known base pairing between P4-P6 and P3-P9. This suggests that the domains interact through multiple tertiary contacts, the nature of which can now be explored in this system. For example, a circularly permuted version of the P4-P6 domain, which folds similarly to the native P4-P6 molecule, formed a stable but inactive complex. Interestingly, activity was demonstrated with the permuted molecule when nucleotides proposed to form a triple-strand interaction with P4 and P6 were restored as part of the P1-P3 substrate or as part of the P3-P9 RNA. Thus, beyond stabilization of the P4-P6 domain, the triple-strand region may facilitate correct orientation of the RNA domains or participate more directly in catalysis.

The existence of RNA enzymes that catalyze phosphodiester transfer reactions suggests that RNA-catalyzed RNA replication might be possible. Indeed, it has been shown that the Tetrahymena and sunY self-splicing introns will catalyze the template-directed ligation of RNA oligonucleotides (Doudna et al., 1989, 1991). We have sought to develop a more general RNA replication system in which arbitrary template sequences could be copied by the assembly of a limited set of short oligonucleotides. Here we examine the use of tetranucleotides as substrates for a primer-extension reaction in which the 5'-nucleoside of the tetranucleotide is the leaving group, and the primer is extended by the remaining three nucleotides. When the 5'-nucleoside is guanosine, the reaction is quite efficient, but a number of competing side reactions occur, in which inappropriate guanosine residues in the primer or the template occupy the guanosine binding site of the ribozyme. We have blocked these side reactions by using the guanosine analogue 2-aminopurine riboside (2AP) as the leaving group, in a reaction catalyzed by a mutant sunY ribozyme that binds efficiently to 2AP and not to guanosine. We have begun to address the issue of the fidelity of the primer-extension reaction by measuring reaction rates with a number of different triplet/template combinations. Our results provide the basis for the further development of an RNA-catalyzed RNA replication system using short oligonucleotide substrates with novel leaving groups.

The Tetrahymena ribozyme has been shown to catalyze an RNA polymerase-like reaction in which an RNA primer is extended by the sequential addition of pN nucleotides derived from GpN dinucleotides, where N = A, C, or U. Here, we show that this reaction is influenced by the presence of a template; bases that can form Watson-Crick base pairs with a template add as much as 25-fold more efficiently than mismatched bases. A mutant enzyme with an altered guanosine binding site can catalyze template-directed primer extension with all four bases when supplied with dinucleotides of the form 2-aminopurine-pN.

Derivatives of the sunY self-splicing intron efficiently catalyzed the synthesis of complementary strand RNA by template-directed assembly of oligonucleotides. These ribozymes were separated into three short RNA fragments that formed active catalytic complexes. One of the multisubunit sunY derivatives catalyzed the synthesis of a strand of RNA complementary to one of its own subunits. These results suggest that prebiotically synthesized oligonucleotides might have been able to assemble into a complex capable of self-replication.

The chemical synthesis of a fully protected ribonucleoside phosphoramidite, containing 2-aminopurine as the base component, and its incorporation into short oligoribonucleotides as substrates for an engineered ribozyme from Tetrahymena is described.

We have constructed all single base substitutions in almost all of the highly conserved residues of the Tetrahymena self-splicing intron. Mutation of highly conserved residues almost invariably leads to loss of enzymatic activity. In many cases, activity could be regained by making additional mutations that restored predicted base-pairings; these second site suppressors in general confirm the secondary structure derived from phylogenetic data. At several positions, our suppression data can be most readily explained by assuming non-Watson-Crick base-pairings. In addition to the requirements imposed by the secondary structure, the sequence of the intron is constrained by "negative interactions", the exclusion of particular nucleotide sequences that would form undesirable secondary structures. A comparison of genetic and phylogenetic data suggests sites that may be involved in tertiary structural interactions.

The Tetrahymena ribozyme can splice together multiple oligonucleotides aligned on a template strand to yield a fully complementary product strand. This reaction demonstrates the feasibility of RNA-catalysed RNA replications.

The group I self-splicing introns act at exon-intron junctions without recognizing a particular sequence. In order to understand splice-site selection, we have developed an assay system based on the Tetrahymena ribozyme to allow the study of numerous 5'-splice-site variants. Cleavage at the correct site requires formation of the correct secondary structure and occurs most efficiently within a 3-base-pair window centered on base pair 5 from the bottom of the P1 stem. Within this window the ribozyme recognizes and cleaves at a "wobble" base pair; the base pair above the cleavage site also influences splicing efficiency. The recognition of RNA structure rather than sequence explains the ability of these transposable introns to splice out of a variety of sequence contexts.

The group I intron from Tetrahymena catalyzes phosphodiester transfer reactions on various RNA substrates. A modified RNA substrate with a phosphorothioate group in one stereoisomeric form at the site of reaction was synthesized in order to determine the stereochemical course of an RNA-catalyzed reaction. The reaction product was digested with a stereospecific nuclease to determine the configuration of the product phosphorothioate. The reaction occurs with inversion of configuration at phosphorus, implying an in-line pathway for the reaction.