Like many structured RNAs, the Tetrahymena group I ribozyme is prone to misfolding. Here we probe a long-lived misfolded species, referred to as M, and uncover paradoxical aspects of its structure and folding. Previous work indicated that a non-native local secondary structure, termed alt P3, led to formation of M during folding in vitro. Surprisingly, hydroxyl radical footprinting, fluorescence measurements with site-specifically incorporated 2-aminopurine, and functional assays indicate that the native P3, not alt P3, is present in the M state. The paradoxical behavior of alt P3 presumably arises because alt P3 biases folding toward M, but, after commitment to this folding pathway and before formation of M, alt P3 is replaced by P3. Further, structural and functional probes demonstrate that the misfolded ribozyme contains extensive native structure, with only local differences between the two states, and the misfolded structure even possesses partial catalytic activity. Despite the similarity of these structures, re-folding of M to the native state is very slow and is strongly accelerated by urea, Na + , and increased temperature and strongly impeded by Mg 2+ and the presence of native peripheral contacts. The paradoxical observations of extensive native structure within the misfolded species but slow conversion of this species to the native state are readily reconciled by a model in which the misfolded state is a topological isomer of the native state, and computational results support the feasibility of this model. We speculate that the complex topology of RNA secondary structures and the inherent rigidity of RNA helices render kinetic traps due to topological isomers considerably more common for RNA than for proteins.

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Phylogenetic comparisons and site-directed mutagenesis indicate that group I introns are composed of a catalytic core that is universally conserved and peripheral elements that are conserved only within intron subclasses. Despite this low overall conservation, peripheral elements are essential for efficient splicing of their parent introns. We have undertaken an in-depth structure-function analysis to investigate the role of one of these elements, P5abc, using the well-characterized ribozyme derived from the Tetrahymena group I intron. Structural comparisons using solution-based free radical cleavage revealed that a ribozyme lacking P5abc (E ∆P5abc ) and E ∆P5abc with P5abc added in trans (E ∆P5abc ‚P5abc) adopt a similar global tertiary structure at Mg 2+ concentrations greater than 20 mM [Doherty, E. A., et al. (1999) Biochemistry 38, 2982-90]. However, free E ∆P5abc is greatly compromised in overall oligonucleotide cleavage activity, even at Mg 2+ concentrations as high as 100 mM. Further characterization of E ∆P5abc via DMS modification revealed local structural differences at several positions in the conserved core that cluster around the substrate binding sites. Kinetic and thermodynamic dissection of individual reaction steps identified defects in binding of both substrates to E ∆P5abc , with g25-fold weaker binding of a guanosine nucleophile and g350-fold weaker docking of the oligonucleotide substrate into its tertiary interactions with the ribozyme core. These defects in binding of the substrates account for essentially all of the 10 4 -fold decrease in overall activity of the deletion mutant. Together, the structural and functional observations suggest that the P5abc peripheral element not only provides stability but also positions active site residues through indirect interactions, thereby preferentially stabilizing the active ribozyme structure relative to alternative less active states. This is consistent with the view that peripheral elements engage in a network of mutually reinforcing interactions that together ensure cooperative folding of the ribozyme to its active structure.Catalytic RNAs can provide rate enhancements that rival those of protein enzymes (1-3). To accomplish this, an RNA must contain sufficient information in its primary sequence to meet two challenges: it must be able to form stable tertiary structure, and it must be able to stabilize the active structure relative to alternate inactive and less active conformations. Relative to proteins, RNA is expected to encounter structural difficulties arising from the charged nature and greater rotational freedom of the phosphodiester backbone, the limited number of nucleoside bases, and the sequestration of the bases by base-pairing within secondary structure (4, 5). We have focused on group I introns to study the means by which RNA molecules can achieve functional structures.The wealth of functional and structural information available for group I introns makes them a powerful system in which to examine the structural underpin...