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While current group I ribozymes use several distinct strategies to function under conditions of low Mg2+ concentration (≤ 3 mM), a deletion mutant of the Tetrahymena ribozyme (ΔP5 ribozyme) is virt...

Ribozymes with modular structures are attractive platforms for the construction of nanoscale RNA objects with biological functions. We designed group I ribozyme dimers as unit ribozyme dimers (Urds), which self-assembled to form their polymeric states and also oligomeric states with defined numbers of Urds. Assembly of Urds yielded catalytic ability of a pair of distinct ribozyme units to cleave two distinct substrates. The morphologies of the assembled ribozyme structures were observed directly by atomic force microscopy (AFM).

In the RNA world, enrichment of self-replicating RNAs would have been beneficial to their survival, amplification, and evolution. Self-assembly of RNAs may be a strategy by which they enrich themselves. We examined the effects of molecular crowding on the activity of a bimolecular group I ribozyme and its derivative that self-assembles to form ribozyme oligomers. In a comparative activity assay using PEG as a molecular crowder, PEG rescued mutations in the parent bimolecular ribozyme more effectively than those in the oligomeric form.

Ribozymes are catalytic RNAs that are attractive platforms for the construction of nanoscale objects with biological functions. We designed a dimeric form of the Tetrahymena group I ribozyme as a unit structure in which two ribozymes were connected in a tail-to-tail manner with a linker element. We introduced a kink-turn motif as a bent linker element of the ribozyme dimer to design a closed trimer with a triangular shape. The oligomeric states of the resulting ribozyme dimers (kUrds) were analyzed biochemically and observed directly by atomic force microscopy (AFM). Formation of kUrd oligomers also triggered trans-splicing reactions, which could be monitored with a reporter system to yield a fluorescent RNA aptamer as the trans-splicing product.

Among cationic molecules that can modulate ribozyme activities, polyamines act as both activator and inhibitor of ribozyme reactions partly due to their structural flexibility. Restriction of structural flexibility of polyamines may allow them to emphasize particular modulation effects. We examined eight stereoisomers of a synthetic pentamine bearing three cyclopentane rings. In the reaction of a structurally unstable group I ribozyme, three stereoisomers exhibited distinct effects as inhibitor, an additive with a neutral effect, and also as an activator.

The emergence of cellular compartmentalization was a crucial step in the hypothetical RNA world and its evolution because it would not only prevent the extinction of RNA self-replication systems due to dispersion/diffusion of their components but also facilitate ribozyme reactions by molecular crowding effects. Here, we proposed and examined self-assembly of RNA components as a primitive cellular-like environment, which may have the ability to mimic cellular compartmentalization and crowding effects. We engineered a bimolecular group I ribozyme to form a one-dimensional (1D)-ribozyme assembly. In the 1D assembly form, severe mutations that inactivated the parent bimolecular ribozyme were modestly rescued resulting in weak catalytic ability.

Bimolecular ribozymes derived by physical dissection of unimolecular ribozymes consisting of two structural modules are promising platforms for the design and construction of assembled RNA nanostructures. Unit RNAs to be assembled intermolecularly into one-dimensional (1D) oligomers are designed by reconnecting the two structural modules in a manner different from the parent ribozymes. This strategy was applied to the Tetrahymena group I ribozyme. We constructed 1D ribozyme oligomers the assembly of which was observed by atomic force microscopy (AFM) and also controlled rationally to design a heterooctamer by differentiating the interface between the two modules.

Self-splicing of group I intron from the 26S rRNA of Candida albicans is essential for maturation of the host RNA. Here, we demonstrated that the co-transcriptional splicing of the intron in vitro was blocked by antisense oligonucleotides (AONs) targeting the P3–P7 core of the intron. The core-targeted AON effectively and specifically inhibited the intron splicing from its host RNA in living C. albicans. Furthermore, flow cytometry experiments showed that the growth inhibition was caused by a fungicidal effect. For the first time, we showed that an AON targeting the ribozyme core folding specifically inhibits the endogenous ribozyme splicing in living cells and specifically kills the intron-containing fungal strains, which sheds light on the development of antifungal drugs in the future.

Joon-Hwa LeeDepartment of Chemistry, Research Institute of Natural Science, and Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju, Gyeongnam 660-701, Korea. E-mail: joonhwa@gnu.ac.krReceived July 31, 2008The P1 duplex of Tetrahymena group I ribozyme is the important system for studying the conformationalchanges in folding of ribozyme. The formation of the P1 duplex between IGS and substrate RNA and thecatalytic activity of ribozyme require a variety of metal ions such as Mg

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 Mg2+ 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.

Background: The Tetrahymena group I ribozyme folds into a complex three-dimensional structure for performing catalytic reactions. The catalysis depends on its catalytic core consisting of two helical domains, P4–P6 and P3–P7, connected by single stranded regions. In the folding process, most of this ribozyme folds in a hierarchical manner in which a kinetically stable intermediate determines the overall folding rate. Results: Although the nature of this intermediate has not yet been elucidated, a mispaired P3 stem (alt-P3) appears a likely candidate. To examine the effects of the alt-P3 structure on the kinetic and thermodynamic properties of the active structure of the ribozyme or its P3–P7 domain formation, we prepared and analysed variant ribozymes in which relative stabilities of the original P3 and alt-P3 structure were altered systematically. Conclusion: The results indicate that the alt-P3 structure is not the major rate-limiting factor in the folding process.

Fluorescence resonance transfer (FRET) was applied for real time monitoring of ribozyme reactions. Group I ribozyme ligation was followed with two separate, fluorescent-labeled RNA substrates. For hammerhead ribozyme cleavage, a double-fluorescent-labeled substrate was used. For the first time we analyzed multiple turnover conditions. Real time monitoring permits convenient analysis of ribozyme kinetics and the sequence-specific, quantitative detection of RNAs in femtomole amounts.

Rigid spinning: Site-directed spin-labeling studies using a rigid nitroxide spin label (C) reveal that both length and sequence of a single-stranded junction (J1/2) modulate nanosecond motional ordering of the substrate-recognition duplex (P1) of the 120 kD group I ribozyme. The studies demonstrate an approach for experimental measurements of nanosecond dynamics in high-molecular-weight RNA complexes.

Crowder molecules in solution alter the configuration potential energy landscape of biological macromolecules. It is therefore critical to account for the influence of these other molecules when describing the folding of RNA inside the cell. Small angle x-ray scattering experiments were used to measure folding of a 64 kDa bacterial group I ribozyme in the presence of polyethylene-glycol with different molecular weights. We find that crowder molecules stabilize more compact states of the unfolded RNA, and also stabilize the folded state with respect to the unfolded state, as measured via the lowering of the folding midpoint on a MgCl2 titration. In addition, stopped-flow SAXS experiments with millisecond resolution show that the addition of crowder molecules speeds up the folding of RNA, even when the stability of the final folded state is held constant. These data indicate that crowder molecules change the folding landscape for RNA, allowing it to fold efficiently in Mg2+ concentrations that are well within the physiological range.

Group I intron ribozymes have a modular architecture and structural elements essential for catalysis. The elements are located in the conserved modular domain P3-P7 that is stabilized by another conserved module, P4-P6. It has been reported that artificial modules can complement the function of the native P4-P6. To exploit the modular architecture of group I ribozyme, we have constructed a hybrid ribozyme by attaching an artificial activator module to the wild-type T4 td ribozyme. Kinetic analysis of the hybrid ribozyme revealed that the artificial module and P4-P6 have unusual positive and negative concerted effects in activating the ribozyme.

Large, structured RNAs traverse folding landscapes in which intermediates and long-lived misfolded states are common. To obtain a comprehensive description of the folding landscape for a structured RNA, it is necessary to understand the connections between productive folding pathways and pathways to these misfolded states. The Tetrahymena group I ribozyme partitions between folding to the native state and to a long-lived misfolded conformation. Here, we show that the observed rate constant for commitment to fold to the native or misfolded states is 1.9 min ˇ1 (37C, 10 mM Mg 2a ), the same within error as the rate constant for overall folding to the native state. Thus, the commitment to alternative folding pathways is made late in the folding process, concomitant with or after the rate-limiting step for overall folding. The ribozyme forms much of its tertiary structure significantly faster than it reaches this commitment point and the tertiary structure is expected to be stable, suggesting that the commitment to fold along pathways to the native or misfolded states is made from a partially structured intermediate. These results allow the misfolded conformation to be incorporated into a folding framework that reconciles previous data and gives quantitative information about the energetic topology of the folding landscape for this RNA. # 2001 Academic Press

Fluorescence polarization has been used recently to monitor diverse macromolecular interactions. In this report, the application of fluorescence polarization has been extended to monitor ribozyme reactions in real time. With fluorescently labeled substrate RNAs, group I ribozyme ligation and hammerhead ribozyme cleavage reactions were studied by fluorescence polarization in substrate excess (multiple turnover) conditions. These results also show that fluorescently labeled RNAs remain active substrates for ribozymes. Furthermore, a direct comparison of fluorescence polarization with fluorescence resonance energy transfer showed that both techniques were comparable for monitoring ribozyme reactions.

Little is known about RNA dynamics, even though it is likely that dynamics are important for both folding and function. The ribozyme, L-21 ScaI, derived from the group I intron of Tetrahymena thermophila (Zaug et al. 1988; Kay and Inoue 1987) provides an excellent system for studying dynamics, since its secondary structure is known (Michel and Dujon 1983; Burke et al. 1987; Cech et al. 1994) and a good model is available for its three-dimensional structure (Michel and Westhof 1990). Moreover, spectroscopic probes have been developed that are sensitive to binding of substrate by this ribozyme (Sugimoto et al. 1989b; Bevilacqua et al. 1992; Kierzek et al. 1993). This permits detection of intermediates and measurement of rate constants for various interconversions. The effects of substitutions and of solution conditions on these rate constants give insights into relationships between structure and dynamics and function. The RNA motion most intensively studied thus far in this system is docking of substrate into the catalytic core of the ribozyme (Bevilacqua et al. 1992, 1993, 1994; Li et al. 1995; Li, Profenno and Turner, unpubl. results). This chapter reviews the methods and results of these studies, and discusses some future perspectives.