Crystal structure of Pistol, a class of self-cleaving ribozyme.

Small self-cleaving ribozymes have been discovered in all evolutionary domains of life. They can catalyze site-specific RNA cleavage, and as a result, they have relevance in gene regulation. Comparative genomic analysis has led to the discovery of a new class of small self-cleaving ribozymes named Pistol. We report the crystal structure of Pistol at 2.97-Å resolution. Our results suggest that the Pistol ribozyme self-cleavage mechanism likely uses a guanine base in the active site pocket to carry out the phosphoester transfer reaction. The guanine G40 is in close proximity to serve as the general base for activating the nucleophile by deprotonating the 2'-hydroxyl to initiate the reaction (phosphoester transfer). Furthermore, G40 can also establish hydrogen bonding interactions with the nonbridging oxygen of the scissile phosphate. The proximity of G32 to the O5' leaving group suggests that G32 may putatively serve as the general acid. The RNA structure of Pistol also contains A-minor interactions, which seem to be important to maintain its tertiary structure and compact fold. Our findings expand the repertoire of ribozyme structures and highlight the conserved evolutionary mechanism used by ribozymes for catalysis.

Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR.

Characterization of the structure and dynamics of nucleic acids by NMR benefits significantly from position specifically labeled nucleotides. Here an E. coli strain deficient in the transketolase gene (tktA) and grown on glucose that is labeled at different carbon sites is shown to facilitate cost-effective and large scale production of useful nucleotides. These nucleotides are site specifically labeled in C1' and C5' with minimal scrambling within the ribose ring. To demonstrate the utility of this labeling approach, the new site-specific labeled and the uniformly labeled nucleotides were used to synthesize a 36-nt RNA containing the catalytically essential domain 5 (D5) of the brown algae group II intron self-splicing ribozyme. The D5 RNA was used in binding and relaxation studies probed by NMR spectroscopy. Key nucleotides in the D5 RNA that are implicated in binding Mg(2+) ions are well resolved. As a result, spectra obtained using selectively labeled nucleotides have higher signal-to-noise ratio compared to those obtained using uniformly labeled nucleotides. Thus, compared to the uniformly (13)C/(15)N-labeled nucleotides, these specifically labeled nucleotides eliminate the extensive (13)C-(13)C coupling within the nitrogenous base and ribose ring, give rise to less crowded and more resolved NMR spectra, and accurate relaxation rates without the need for constant-time or band-selective decoupled NMR experiments. These position selective labeled nucleotides should, therefore, find wide use in NMR analysis of biologically interesting RNA molecules.

Protein-facilitated folding of group II intron ribozymes.

Multiple studies hypothesize that DEAD-box proteins facilitate folding of the ai5gamma group II intron. However, these conclusions are generally inferred from splicing kinetics, and not from direct monitoring of DEAD-box protein-facilitated folding of the intron. Using native gel electrophoresis and dimethyl sulfate structural probing, we monitored Mss-116-facilitated folding of ai5gamma intron ribozymes and a catalytically active self-splicing RNA containing full-length intron and short exons. We found that the protein directly stimulates folding of these RNAs by accelerating formation of the compact near-native state. This process occurs in an ATP-independent manner, although ATP is required for the protein turnover. As Mss 116 binds RNA nonspecifically, most binding events do not result in the formation of the compact state, and ATP is required for the protein to dissociate from such nonproductive complexes and rebind the unfolded RNA. Results obtained from experiments at different concentrations of magnesium ions suggest that Mss 116 stimulates folding of ai5gamma ribozymes by promoting the formation of unstable folding intermediates, which is then followed by a cascade of folding events resulting in the formation of the compact near-native state. Dimethyl sulfate probing results suggest that the compact state formed in the presence of the protein is identical to the near-native state formed more slowly in its absence. Our results also indicate that Mss 116 does not stabilize the native state of the ribozyme, but that such stabilization results from binding of attached exons.

Single-molecule studies of group II intron ribozymes.

Group II intron ribozymes fold into their native structure by a unique stepwise process that involves an initial slow compaction followed by fast formation of the native state in a Mg(2+)-dependent manner. Single-molecule fluorescence reveals three distinct on-pathway conformations in dynamic equilibrium connected by relatively small activation barriers. From a most stable near-native state, the unobserved catalytically active conformer is reached. This most compact conformer occurs only transiently above 20 mM Mg(2+) and is stabilized by substrate binding, which together explain the slow cleavage of the ribozyme. Structural dynamics increase with increasing Mg(2+) concentrations, enabling the enzyme to reach its active state.

A kinetic intermediate that regulates proper folding of a group II intron RNA.

The D135 group II intron ribozyme follows a unique folding pathway that is direct and appears to be devoid of kinetic traps. During the earliest stages of folding, D135 collapses slowly to a compact intermediate, and all subsequent assembly events are rapid. Collapse of intron domain 1 (D1) has been shown to limit the rate constant for D135 folding, although the specific substructure of the D1 kinetic intermediate has not yet been identified. Employing time-resolved nucleotide analog interference mapping, we have identified a cluster of atoms within the D1 main stem that control the rate constant for D135 collapse. Functional groups within the kappa-zeta element are particularly important for this earliest stage of folding, which is intriguing given that this same motif also serves later as the docking site for catalytic domain 5. More important, the kappa-zeta element is shown to be a divalent ion binding pocket, indicating that this region is a Mg(2+)-dependent switch that initiates the cascade of D135 folding events. By measuring the Mg(2+) dependence of the compaction rate constant, we conclude that the actual rate-limiting step in D1 compaction involves the formation of an unstable folding intermediate that is captured by the binding of Mg(2+). This carefully orchestrated folding pathway, in which formation of an active-site docking region is early and rate limiting, ensures proper folding of the intron core and faithful splicing. It may represent an important paradigm for the folding of large, multidomain RNA molecules.

Solution structure of domain 6 from a self-splicing group II intron ribozyme: a Mg(2+) binding site is located close to the stacked branch adenosine.

Group II intron self-splicing is essential for the correct expression of organellar genes in plants, fungi, and yeast, as well as of bacterial genes. Self-excision of these autocatalytic introns from the primary RNA transcript is achieved in a two-step mechanism that is apparently analogous to that of the eukaryotic spliceosome. The 2'-OH of a conserved adenosine (the branch point) located within domain 6 (D6) acts as the nucleophile in the first step of splicing. Despite the biological importance of group II introns, little is known about their structural organization and usage of metal ions in catalysis. Here we report the first solution structure of a catalytically active D6 construct encompassing the branch point and the neighboring helical regions from the mitochondrial yeast intron ai5gamma. The branch adenosine is the single unpaired nucleotide, and, in contrast to the spliceosomal branch site, resides within the helix, being partially stacked between two flanking GU wobble pairs. We identified a novel prominent Mg(2+) binding site in the major groove of the branch site. Importantly, Mg(2+) addition does not impair the stacking of the branch adenosine, rather it strengthens the interaction with the flanking uridines, as shown by NMR and fluorescence studies. This means that domain 6 presents the branch adenosine in a stacked fashion to the core of group II introns upon folding to the active conformation.

Group II intron folding under near-physiological conditions: collapsing to the near-native state.

The folding of group II intron ribozymes has been studied extensively under optimal conditions for self-splicing in vitro (42 degrees C and high magnesium ion concentrations). In these cases, the ribozymes fold directly to the native state by an apparent two-state mechanism involving the formation of an obligate intermediate within intron domain 1. We have now characterized the folding pathway under near-physiological conditions. We observe that compaction of the RNA proceeds slowly to completion, even at low magnesium concentration (3 mM). Kinetic analysis shows that this compact species is a "near-native" intermediate state that is readily chased into the native state by the addition of high salt. Structural probing reveals that the near-native state represents a compact domain 1 scaffold that is not yet docked with the catalytic domains (D3 and D5). Interestingly, native ribozyme reverts to the near-native state upon reduction in magnesium concentration. Therefore, while the intron can sustain the intermediate state under physiological conditions, the native structure is not maintained and is likely to require stabilization by protein cofactors in vivo.

A folding control element for tertiary collapse of a group II intron ribozyme.

Ribozymes derived from the group II intron ai5gamma collapse to a compact intermediate, folding to the native state through a slow, direct pathway that is unperturbed by kinetic traps. Molecular collapse of ribozyme D135 requires high magnesium concentrations and is thought to involve a structural element in domain 1 (D1). We used nucleotide analog interference mapping, in combination with nondenaturing gel electrophoresis, to identify RNA substructures and functional groups that are essential for D135 tertiary collapse. This revealed that the most crucial atoms for compaction are located within a small section of D1 that includes the kappa and zeta elements. This small substructure controls specific collapse of the molecule and, in later steps of the folding pathway, it forms the docking site for catalytic D5. In this way, the stage is set for proper active site formation during the earliest steps of ribozyme folding.

Conformation of the Group II intron branch site in solution.

Group II introns are multidomain ribozymes that catalyze their own removal from pre-mRNA. The nucleophile for the first cleavage step is the 2'OH of a specific adenosine within domain 6 (D6), called the branch site. Mechanistic parallels and limited secondary structural similarity with the eukaryotic spliceosome lead many to speculate that the two systems have a common ancestry. We have elucidated structural features of the branch site region and the importance of the internal loop to branch site conformation within D6 of the ai5gamma Group II intron by NMR and fluorescence spectroscopy. Fluorescence experiments in which 2-aminopurine was substituted for the branch site adenosine suggest that the branch site base is exposed to solvent and that this position is enhanced by Mg2+ or Ca2+. Upfield NMR chemical shifts of imino protons of the two uridine residues flanking the branch site adenosine, and an n --> n + 2 NOE between them, suggest a stacked intrahelical conformation of the two uridines. In contrast, results of NMR and 2-aminopurine fluorescence spectra of a mutated D6 from which the internal loop had been deleted suggest a less exposed position of the branch site adenosine, which is likely to form a G-A base pair with the opposing 3'G. These findings describe a model in which the branch site adenosine of D6 is in an extrahelical position, surrounded by two intrahelical bases. The internal loop and divalent metal ions facilitate this motif.

An obligate intermediate along the slow folding pathway of a group II intron ribozyme.

Most RNA molecules collapse rapidly and reach the native state through a pathway that contains numerous traps and unproductive intermediates. The D135 group II intron ribozyme is unusual in that it can fold slowly and directly to the native state, despite its large size and structural complexity. Here we use hydroxyl radical footprinting and native gel analysis to monitor the timescale of tertiary structure collapse and to detect the presence of obligate intermediates along the folding pathway of D135. We find that structural collapse and native folding of Domain 1 precede assembly of the entire ribozyme, indicating that D1 contains an on-pathway intermediate to folding of the D135 ribozyme. Subsequent docking of Domains 3 and 5, for which D1 provides a preorganized scaffold, appears to be very fast and independent of one another. In contrast to other RNAs, the D135 ribozyme undergoes slow tertiary collapse to a compacted state, with a rate constant that is also limited by the formation D1. These findings provide a new paradigm for RNA folding and they underscore the diversity of RNA biophysical behaviors.

Group I aptazymes as genetic regulatory switches.

BACKGROUND: Allosteric ribozymes (aptazymes) that have extraordinary activation parameters have been generated in vitro by design and selection. For example, hammerhead and ligase ribozymes that are activated by small organic effectors and protein effectors have been selected from random sequence pools appended to extant ribozymes. Many ribozymes, especially self-splicing introns, are known control gene regulation or viral replication in vivo. We attempted to generate Group I self-splicing introns that were activated by a small organic effector, theophylline, and to show that such Group I aptazymes could mediate theophylline-dependent splicing in vivo. RESULTS: By appending aptamers to the Group I self-splicing intron, we have generated a Group I aptazyme whose in vivo splicing is controlled by exogenously added small molecules. Substantial differences in gene regulation could be observed with compounds that differed by as little as a single methyl group. The effector-specificity of the Group I aptazyme could be rationally engineered for new effector molecules. CONCLUSION: Group I aptazymes may find applications as genetic regulatory switches for generating conditional knockouts at the level of mRNA or for developing economically viable gene therapies.