Time-resolved NMR spectroscopy was applied to study ribozyme-mediated RNA catalysis in a mutant of the hairpin ribozyme, the adenine-dependent hairpin ribozyme (ADHR; M. Meli, et al. J. Biol. Chem. 2003, 278, 9835-9842) with atomic resolution. The mutant ADHR was designed to investigate the role of cofactors in RNA catalytic mechanisms in order to understand cellular processes that could have been present in the archaic "RNA world" and of their evolution towards functional RNAs in modern cellular processes, as for example, found in the glmS ribozyme. Conformational changes due to RNA cleavage were analyzed following spectral changes of the NMR imino proton resonances that could be assigned both for the pre- and postcleaved conformation for this 80-nucleotide long RNA.(31)P NMR spectroscopic studies allowed us to confirm the formation of a cyclic phosphodiester as a result of the cleavage process. For ADHR, both metal ions and the cofactor adenine are essential for self-cleaving activity. The interaction of the ribozyme with the cofactor adenine is found to be transient and too weak to significantly change the RNA structure or to modulate the spectroscopic characteristics of the cofactor. ADHR therefore represents a ribozyme in which high activation barriers have to be overcome to populate cleavage-competent states that exhibit short life times. We show that conformational dynamics, but not the chemistry, constitute the rate-limiting step in catalysis of the adenine-dependent hairpin ribozyme.

The existence of an "RNA world" as an early step in the history of life increases the interest for the characterization of these biomolecules. The hairpin ribozyme studied here is a self-cleaving/ligating motif found in the minus strand of the satellite RNA associated with Tobacco ringspot virus. Surface enhanced Raman spectroscopy (SERS) is a powerful tool to study trace amounts of RNA. In controlled conditions, a SERS signal is proportional to the amount of free residues adsorbed on the metal surface. Upon RNA cleavage, residues are unpaired and free to interact with metal. SERS procedures are used to monitor and quantify the catalysis of ribozyme cleavage at biological concentrations in real time; thus they propose an interesting alternative to electrophoretic methods.(c) 2009 Wiley Periodicals, Inc. Biopolymers, 2009.

The dynamic mechanisms by which RNAs acquire biologically functional structures are of increasing importance to the rapidly expanding fields of RNA therapeutics and biotechnology. Large energy barriers separating misfolded and functional states arising from alternate base pairing are a well-appreciated characteristic of RNA. In contrast, it is typically assumed that functionally folded RNA occupies a single native basin of attraction that is free of deeply dividing energy barriers (ergodic hypothesis). This assumption is widely used as an implicit basis to interpret experimental ensemble-averaged data. Here, we develop an experimental approach to isolate persistent sub-populations of a small RNA enzyme and show by single molecule fluorescence resonance energy transfer (smFRET), biochemical probing and high-resolution mass spectrometry that commitment to one of several catalytically active folds occurs unexpectedly high on the RNA folding energy landscape, resulting in partially irreversible folding. Our experiments reveal the retention of molecular heterogeneity following the complete loss of all native secondary and tertiary structure. Our results demonstrate a surprising longevity of molecular heterogeneity and advance our current understanding beyond that of non-functional misfolds of RNA kinetically trapped on a rugged folding-free energy landscape.

Non-coding RNAs of complex tertiary structure are involved in numerous aspects of the replication and processing of genetic information in many organisms; however, an understanding of the complex relationship between their structural dynamics and function is only slowly emerging. The Neurospora Varkud Satellite (VS) ribozyme provides a model system to address this relationship. First, it adopts a tertiary structure assembled from common elements, a kissing loop and two three-way junctions. Second, catalytic activity of the ribozyme is essential for replication of VS RNA in vivo and can be readily assayed in vitro. Here we exploit single molecule FRET to show that the VS ribozyme exhibits previously unobserved dynamic and heterogeneous hierarchical folding into an active structure. Readily reversible kissing loop formation combined with slow cleavage of the upstream substrate helix suggests a model whereby the structural dynamics of the VS ribozyme favor cleavage of the substrate downstream of the ribozyme core instead. This preference is expected to facilitate processing of the multimeric RNA replication intermediate into circular VS RNA, which is the predominant form observed in vivo.

The hairpin ribozyme acts as a reversible, site-specific endoribonuclease that ligates much more rapidly than it cleaves cognate substrate. While the reaction pathway for ligation is the reversal of cleavage, little is known about the atomic and electrostatic details of the two processes. Here, we report the functional consequences of molecular substitutions of A9 and A10, two highly conserved nucleobases located adjacent to the hairpin ribozyme active site, using G, C, U, 2-aminopurine, 2,6-diaminopurine, purine, and inosine. Cleavage and ligation kinetics were analyzed, tertiary folding was monitored by hydroxyl radical footprinting, and interdomain docking was studied by native gel electrophoresis. We determined that nucleobase substitutions that exhibit significant levels of interference with tertiary folding and interdomain docking have relatively large inhibitory effects on ligation rates while showing little inhibition of cleavage. Indeed, one variant, A10G, showed a fivefold enhancement of cleavage rate and no detectable ligation, and we suggest that this property may be uniquely well suited to intracellular targeted RNA cleavage applications. Results support a model in which formation of a kinetically stable tertiary structure is essential for ligation of the hairpin ribozyme, but is not necessary for cleavage.

The ability of RNA to catalyze chemical reactions was first demonstrated 25 years ago with the discovery that group I introns and RNase P function as RNA enzymes (ribozymes). Several additional ribozymes were subsequently identified, most notably the ribosome, followed by intense mechanistic studies. More recently, the introduction of single molecule tools has dissected the kinetic steps of several ribozymes in unprecedented detail and has revealed surprising heterogeneity not evident from ensemble approaches. Still, many fundamental questions of how RNA enzymes work at the molecular level remain unanswered. This review surveys the current status of our understanding of RNA catalysis at the single molecule level and discusses the existing challenges and opportunities in developing suitable assays.(c) 2007 Wiley Periodicals, Inc. Biopolymers, 2007.

Single-molecule FRET is a powerful tool for probing the kinetic mechanism of a complex enzymatic reaction. However, not every reaction intermediate can be identified via a distinct FRET value, making it difficult to fully dissect a multistep reaction pathway. Here, we demonstrate a method using sequential kinetic experiments to differentiate each reaction intermediate by a distinct time sequence of FRET signal (a kinetic "fingerprint"). Our model system, the two-way junction hairpin ribozyme, catalyzes a multistep reversible RNA cleavage reaction, which comprises two structural transition steps (docking/undocking) and one chemical reaction step (cleavage/ligation). Whereas the docked and undocked forms of the enzyme display distinct FRET values, the cleaved and ligated forms do not. To overcome this difficulty, we used Mg(2+) pulse-chase experiments to differentiate each reaction intermediate by a distinct kinetic fingerprint at the single-molecule level. This method allowed us to unambiguously determine the rate constant of each reaction step and fully characterize the reaction pathway by using the chemically competent enzyme-substrate complex. We found that the ligated form of the enzyme highly favors the docked state, whereas undocking becomes accelerated upon cleavage by two orders of magnitude, a result different from that obtained with chemically blocked substrate and product analogs. The overall cleavage reaction is rate-limited by the docking/undocking kinetics and the internal cleavage/ligation equilibrium, contrasting the rate-limiting mechanism of the four-way junction ribozyme. These results underscore the kinetic interdependence of reversible steps on an enzymatic reaction pathway and demonstrate a potentially general route to dissect them.

The hairpin ribozyme is a small catalytic motif found in plant satellite RNAs where it catalyzes a reversible self-cleavage reaction during processing of replication intermediates. Crystallographic studies of hairpin ribozymes have provided high-resolution views of the RNA functional groups that comprise the active site and stimulated biochemical studies that probed the contributions of nucleobase functional groups to catalytic chemistry. The dramatic loss of activity that results from perturbation of active site architecture points to the importance of positioning and orientation in catalytic rate acceleration. The current study focuses on the network of noncovalent interactions that align nucleophilic and leaving group oxygens in the orientation required for the SN2-type reaction mechanism and orient the active site nucleobases near the reactive phosphate to facilitate catalytic chemistry. Nucleotide modifications that alter or eliminate individual hydrogen bonding partners had different effects on the activation barrier to catalysis, the stability of ribozyme complexes in the ground state, and the internal equilibrium between cleavage and ligation of bound products. Furthermore, substitution of hydrogen bond donors and acceptors with seemingly equivalent pairs sometimes had very different functional consequences. These biochemical analyses augment high-resolution structural information to provide insights into the functional significance of active site architecture.

Hepatitis C virus (HCV), the major etiological agent of transfusion-associated non-A, non-B hepatitis, is a severe health problem affecting up to 3% of the world population. Since its identification in 1989, enormous efforts have been made to characterize the viral cycle. However, many details regarding the virus' penetration of hepatocytes, its replication and translation, and the assembling of virions remain unknown, mostly because of a lack of an efficient culture system. This has also hampered the development of fully effective antiviral drugs. Current treatments based on the combination of interferon and ribavirin trigger a sustained virological response in only 40% of infected individuals, thus the development of alternative therapeutic strategies is a major research goal. Nucleic acid based therapeutic agents may be of some potential in hepatitis C treatment. In recent years, much effort has gone into the improvement of DNA and RNA molecules as specific gene silencing tools. This review summarizes the state of the art in the development of new HCV therapies, paying special attention to those involving antisense oligonucleotides, aptamers, ribozymes, decoys and siRNA inhibitors. The identification of potential viral targets is also discussed.

The hairpin ribozyme is a small endonucleolytic RNA motif with potential for targeted RNA inactivation. It optimally cleaves substrates containing the sequence 5'-GU-3' immediately 5' of G. Previously, we have shown that tertiary structure docking of its two domains is an essential step in the reaction pathway of the hairpin ribozyme. Here we show, combining biochemical and fluorescence structure and function probing techniques, that any mutation of the substrate base U leads to a docked RNA fold, yet decreases cleavage activity. The docked mutant complex shares with the wild-type complex a common interdomain distance as measured by time-resolved fluorescence resonance energy transfer (FRET) as well as the same solvent-inaccessible core as detected by hydroxyl-radical protection; hence, the mutant complex appears nativelike. FRET experiments also indicate that mutant docking is kinetically more complex, yet with an equilibrium shifted toward the docked conformation. Using 2-aminopurine as a site-specific fluorescent probe in place of the wild-type U, a local structural rearrangement in the substrate is observed. This substrate straining accompanies global domain docking and involves unstacking of the base and restriction of its conformational dynamics, as detected by time-resolved 2-aminopurine fluorescence spectroscopy. These data appear to invoke a mechanism of functional interference by a single base mutation, in which the ribozyme-substrate complex becomes trapped in a nativelike fold preceding the chemical transition state.

The two domains of the hairpin ribozyme-substrate complex, usually depicted as straight structural elements, must interact with one another in order to form an active conformation. Little is known about the internal geometry of the individual domains in an active docked complex. Using various crosslinking and structural approaches in conjunction with molecular modeling (constraint-satisfaction program MC-SYM), we have investigated the conformation of the substrate-binding domain in the context of the active docked ribozyme-substrate complex. The model generated by MC-SYM showed that the domain is not straight but adopts a bent conformation (D-shaped) in the docked state of the ribozyme, indicating that the two helices bounding the internal loop are closer than was previously assumed. This arrangement rationalizes the observed ability of hairpin ribozymes with a circularized substrate-binding strand to cleave a circular substrate, and provides essential information concerning the organization of the substrate in the active conformation. The internal geometry of the substrate-binding strand places G8 of the substrate-binding strand near the cleavage site, which has allowed us to predict the crucial role played by this nucleotide in the reaction chemistry.

The complex between the hairpin ribozyme and its substrate consists of two domains that must interact in order to form a catalytic complex, yet experimental evidence concerning the points of interaction between the two domains has been lacking. Here, we report the use of hydroxyl radical footprinting to define the interface between the two domains. Cations that support very efficient ribozyme catalysis (magnesium and cobalt(III) hexammine) lead to the formation of a docked complex that features several regions of protection, indicating a solvent-inaccessible core within the tertiary structure of the complex. Cations that are suboptimal in cleavage reactions do not produce complexes with regions of reduced solvent accessibility. Nucleotides encompassing the substrate cleavage site (c-2, a-1, g+1, and u+2) are strongly protected, suggesting their internalization into the catalytic core. Four distinct segments of the ribozyme are protected, including G11-A14, C25-C27, A38, and U42-A43. Protection of these sites is eliminated when g+1, an essential base at the cleavage site, is replaced by A. In addition, mutations which are known to decrease the fraction of docked complexes decrease or eliminate formation of a solvent-inaccessible core. Taken together, these observations demonstrate that we have identified the catalytic core of the active hairpin ribozyme-substrate complex.

The catalytic determinants for the cleavage and ligation reactions mediated by the hairpin ribozyme are integral to the polyribonucleotide chain. We describe experiments that place G8, a critical guanosine, at the active site, and point to an essential role in catalysis. Cross-linking and modeling show that formation of a catalytic complex is accompanied by a conformational change in which N1 and O6 of G8 become closely apposed to the scissile phosphodiester. UV cross-linking, hydroxyl-radical footprinting and native gel electrophoresis indicate that G8 variants inhibit the reaction at a step following domain association, and that the tertiary structure of the inactive complex is not measurably altered. Rate-pH profiles and fluorescence spectroscopy show that protonation at the N1 position of G8 is required for catalysis, and that modification of O6 can inhibit the reaction. Kinetic solvent isotope analysis suggests that two protons are transferred during the rate-limiting step, consistent with rate-limiting cleavage chemistry involving concerted deprotonation of the attacking 2'-OH and protonation of the 5'-O leaving group. We propose mechanistic models that are consistent with these data, including some that invoke a novel keto-enol tautomerization.

The hairpin ribozyme is a short endonucleolytic RNA motif isolated from a family of related plant virus satellite RNAs. It consists of two independently folding domains, each comprising two Watson-Crick helices flanking a conserved internal loop. The domains need to physically interact (dock) for catalysis of site-specific cleavage and ligation reactions. Using tapping-mode atomic force microscopy in aqueous buffer solution, we were able to produce high quality images of individual hairpin ribozyme molecules with extended terminal helices. Three RNA constructs with either the essential cleavage site guanosine or a detrimental adenosine substitution and with or without a 6-nt insertion to confer flexibility to the interdomain hinge show structural differences that correlate with their ability to form the active docked conformation. The observed contour lengths and shapes are consistent with previous bulk-solution measurements of the transient electric dichroism decays for the same RNA constructs. The active docked construct appears as an asymmetrically docked conformation that might be an indication of a more complicated docking event than a simple collapse around the interdomain hinge.

The catalysis of site-specific RNA cleavage and ligation by the hairpin ribozyme requires the formation of a tertiary interaction between two independently folded internal loop domains, A and B. Within the B domain, a tertiary structure has been identified, known as the loop E motif, that has been observed in many naturally occurring RNAs. One characteristic of this motif is a partial cross-strand stack of a G residue on a U residue. In a few cases, including loop B of the hairpin ribozyme, this unusual arrangement gives rise to photoreactivity. In the hairpin, G21 and U42 can be UV cross-linked. Here we show that docking of the two domains correlates very strongly with a loss of UV reactivity of these bases. The rate of the loss of photoreactivity during folding is in close agreement with the kinetics of interdomain docking as determined by hydroxyl-radical footprinting and fluorescence resonance energy transfer (FRET). Fixing the structure of the complex in the cross-linked form results in an inability of the two domains to dock and catalyze the cleavage reaction, suggesting that the conformational change is essential for catalysis.

Chemical footprinting methods have been used extensively to probe the structures of biologically important RNAs at nucleotide resolution. One of these methods, hydroxyl-radical footprinting, has recently been employed to study the kinetics of RNA folding. Hydroxyl radicals can be generated by a number of different methods, including Fe(II)-EDTA complexes, synchrotron radiation, and peroxynitrous acid disproportionation. The latter two methods have been used for kinetic studies of RNA folding. We have taken advantage of rapid hydroxyl-radical generation by Fe(II)-EDTA-hydrogen peroxide solutions to develop a benchtop method to study folding kinetics of RNA complexes. This technique can be performed using commercially available chemicals, and can be used to accurately define RNA folding rate constants slower than 6 min(-1). Here we report the method and an example of time-resolved footprinting on the hairpin ribozyme, a small endoribonuclease and RNA ligase.

Catalysis by the hairpin ribozyme is stimulated by a wide range of both simple and complex metallic and organic cations. This independence from divalent metal ion binding unequivocally excludes inner-sphere coordination to RNA as an obligatory role for metal ions in catalysis. Hence, the hairpin ribozyme is a unique model to study the role of outer-sphere coordinated cations in folding of a catalytically functional RNA structure. Here, we demonstrate that micromolar concentrations of a deprotonated aqueous complex of the lanthanide metal ion terbium(III), Tb(OH)(aq)(2+), reversibly inhibit the ribozyme by competing for a crucial, yet non-selective cation binding site. Tb(OH)(aq)(2+) also reports a likely location of this binding site through backbone hydrolysis, and permits the analysis of metal binding through sensitized luminescence. We propose that the critical cation-binding site is located at a position within the catalytic core that displays an appropriately-sized pocket and a high negative charge density. We show that cationic occupancy of this site is required for tertiary folding and catalysis, yet the site can be productively occupied by a wide variety of cations. It is striking that micromolar Tb(OH)(aq)(2+) concentrations are compatible with tertiary folding, yet interfere with catalysis. The motif implicated here in cation-binding has also been found to organize the structure of multi-helix loops in evolutionary ancient ribosomal RNAs. Our findings, therefore, illuminate general principles of non-selective outer-sphere cation binding in RNA structure and function that may have prevailed in primitive ribozymes of an early "RNA world".

This study demonstrates that realizing the correlation between in situ crystallographic structure modifications of an electrochromic material and its functionality leads to improved performances, which can then contribute to a variety of energy-efficient applications.

The 8-17 Deoxyribozyme is an in vitro selected enzyme capable of sequence-specific cleavage of RNA. While selected to be a magnesium and zinc-utilizing DNAzyme, the 8-17 has been shown to efficiently utilize lead for its catalysis. Fluorescence-based experiments have indicated that while the magnesium- and zinc-utilizing versions of the DNAzyme-substrate complex need to form a defined tertiary structure to be active, no such global folding is required for the lead-mediated activity. Herein, we have broadly investigated this phenomenon, including the use of contact photo-crosslinking to map the tertiary fold of the lead-dependent DNAzyme. While our results recapitulate that global folding is not required for the lead activity, they also reveal strikingly distinct lead-mediated modes of activity under conditions of low versus moderate solution ionic strength. Even in very low salt buffers, where no global folding of the 8-17 occurs, the DNAzyme's active site appears to form a distinct local fold, one that cannot easily be modeled by DNA/RNA constructs that preserve key sequence and secondary structure features of the active site.

Attachment of three different fluorophores to an aza-oxa cryptand (L) gives a transition-metal ion induced two-step fluorescence resonance energy transfer system.

The <i>glmS</i> ribozyme is a conserved riboswitch in numerous Gram-positive bacteria and is located upstream of the glucosamine-6-phosphate (GlcN6P) synthetase reading frame. Binding of GlcN6P activates site-specific self-cleavage of the glmS mRNA, resulting in the down regulation of <i>glmS</i> gene expression. Unlike other riboswitches, the <i>glmS</i> ribozyme does not undergo structural rearrangement upon metabolite binding, indicating that the metabolite binding pocket is preformed in the absence of ligand. This observation led us to test if individual steps in the reaction pathway could be dissected by initiating the cleavage reaction before or after Mg²⁺-dependent folding. Here we show that self-cleavage reactions initiated with simultaneous addition of Mg²⁺ and GlcN6P are slow,(3 min^-1), compared to reactions initiated by GlcN6P addition to <i>glmS</i> RNA that has been prefolded in Mg²⁺-containing buffer,(72 min^-1). These data indicate that some level of Mg²⁺-dependent folding is rate limiting for catalysis. Reactions initiated by addition of GlcN6P to prefolded ribozyme also resulted in a 30-fold increase in the apparent ligand K_d compared to reactions initiated by a global folding step. Time-resolved hydroxyl-radical footprinting was employed to determine if global tertiary structure formation is the rate-limiting step. The results of these experiments provided evidence for fast and largely concerted folding of the global tertiary structure,>13 min^-1. This indicates that the rate-limiting step that we have identified is either a slow folding step between the fast initial folding and ligand binding events, or represents the rate of escape from a native-like folding trap.

Active site guanines are critical for self-cleavage reactions of several ribozymes, but their precise functions in catalysis are unclear. To learn whether protonated or deprotonated forms of guanine predominate in the active site, microscopic pKa values were determined for ionization of 8-azaguanosine substituted for G8 in the active site of a fully functional hairpin ribozyme in order to determine microscopic pKa values for 8-azaguanine deprotonation from the pH dependence of fluorescence. Microscopic pKa values above 9 for deprotonation of 8-azaguanine in the active site were about 3 units higher than apparent pKa values determined from the pH dependence of self-cleavage kinetics. Thus, the increase in activity with increasing pH does not correlate with deprotonation of G8, and most of G8 is protonated at neutral pH. These results do not exclude a role in proton transfer, but a simple interpretation is that G8 functions in the protonated form, perhaps by donating hydrogen bonds.

The hairpin ribozyme acts as a reversible, site-specific endoribonuclease that ligates much more rapidly than it cleaves cognate substrate. While the reaction pathway for ligation is the reversal of cleavage, little is known about the atomic and electrostatic details of the two processes. Here, we report the functional consequences of molecular substitutions of A9 and A10, two highly conserved nucleobases located adjacent to the hairpin ribozyme active site, using G, C, U, 2-aminopurine, 2,6-diaminopurine, purine, and inosine. Cleavage and ligation kinetics were analyzed, tertiary folding was monitored by hydroxyl radical footprinting, and interdomain docking was studied by native gel electrophoresis. We determined that nucleobase substitutions that exhibit significant levels of interference with tertiary folding and interdomain docking have relatively large inhibitory effects on ligation rates while showing little inhibition of cleavage. Indeed, one variant, A10G, showed a fivefold enhancement of cleavage rate and no detectable ligation, and we suggest that this property may be uniquely well suited to intracellular targeted RNA cleavage applications. Results support a model in which formation of a kinetically stable tertiary structure is essential for ligation of the hairpin ribozyme, but is not necessary for cleavage.

Protein metalloenzymes use various modes for functions for which metal-dependent global conformational change is required in some cases but not in others. In contrast, most ribozymes require a global folding that almost always precedes enzyme reactions. Herein we studied metal-dependent folding and cleavage activity of the 8-17 DNAzyme using single-molecule fluorescence resonance energy transfer. Addition of Zn(2+) and Mg(2+) induced folding of the DNAzyme into a more compact structure followed by a cleavage reaction, which suggests that the DNAzyme may require metal-dependent global folding for activation. In the presence of Pb(2+), however, the cleavage reaction occurred without a precedent folding step, which suggests that the DNAzyme may be prearranged to accept Pb(2+) for the activity. Neither ligation reaction of the cleaved substrates nor dynamic changes between folded and unfolded states was observed. These features may contribute to the unusually fast Pb(2+)-dependent reaction of the DNAzyme. These results suggest that DNAzymes can use all modes of activation that metalloproteins use.

RNAs fold into complex three-dimensional structures that are critical to their various biological functions. These structures often form independently of the RNA secondary structure, and the three-dimensional fold is stabilized by specific tertiary interactions between various motifs. However, the detailed molecular mechanisms that drive formation of these RNA tertiary interactions are not well understood. The most commonly used variables for probing the thermodynamics or kinetics of RNA-RNA interactions are temperature, metal ion concentration, and chemical denaturant concentration. In this study, the effects of hydrostatic pressure and nondenaturing cosolutes were examined. The commonly occurring GAAA tetraloop-receptor RNA tertiary motif was used as a model system, with formation of this interaction assayed by fluorescence resonance energy transfer. The results showed that the GAAA tetraloop-receptor interaction is slightly destabilized by hydrostatic pressure, and analysis of the pressure data yielded the change in partial molar volume for this RNA-RNA interaction. Polyethylene glycol and dextran cosolutes were both shown to favor formation of the tertiary structure, whereas sucrose and glycerol had little effect on the folding of the RNA. These results demonstrate that hydrostatic pressure and nondenaturing cosolute concentration can be useful variables for investigating tertiary or intermolecular RNA-RNA interactions.

Single-molecule FRET is a powerful tool for probing the kinetic mechanism of a complex enzymatic reaction. However, not every reaction intermediate can be identified via a distinct FRET value, making it difficult to fully dissect a multistep reaction pathway. Here, we demonstrate a method using sequential kinetic experiments to differentiate each reaction intermediate by a distinct time sequence of FRET signal (a kinetic "fingerprint"). Our model system, the two-way junction hairpin ribozyme, catalyzes a multistep reversible RNA cleavage reaction, which comprises two structural transition steps (docking/undocking) and one chemical reaction step (cleavage/ligation). Whereas the docked and undocked forms of the enzyme display distinct FRET values, the cleaved and ligated forms do not. To overcome this difficulty, we used Mg(2+) pulse-chase experiments to differentiate each reaction intermediate by a distinct kinetic fingerprint at the single-molecule level. This method allowed us to unambiguously determine the rate constant of each reaction step and fully characterize the reaction pathway by using the chemically competent enzyme-substrate complex. We found that the ligated form of the enzyme highly favors the docked state, whereas undocking becomes accelerated upon cleavage by two orders of magnitude, a result different from that obtained with chemically blocked substrate and product analogs. The overall cleavage reaction is rate-limited by the docking/undocking kinetics and the internal cleavage/ligation equilibrium, contrasting the rate-limiting mechanism of the four-way junction ribozyme. These results underscore the kinetic interdependence of reversible steps on an enzymatic reaction pathway and demonstrate a potentially general route to dissect them.

High hydrostatic pressure (HHP) technique was used to evaluate a mechanism of RNA hydrolysis with RNA. We showed that hammerhead ribozyme specifically cleaves RNA substrate at HHP in the absence of Mg(2+). A deoxyribozyme "10-23" was active in the same conditions. These results pointed out that the hydrolytic activity of nucleic acid depends on proper tertiary structure of a complex with a substrate. They prove that magnesium ion is not directly involved in catalysis process. On that basis we show the mechanism of RNA hydrolysis catalyzed with nucleic acids at HHP.