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

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

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

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

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

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

The conformational human pathogenic hepatitis delta virus (HDV) employs a unique self-cleaving catalytic RNA motif, the HDV ribozyme, during double-rolling circle replication.Our Fluorescence spectroscopy, circular dichroism, terbium(III) footprinting, and X-ray crystallography of precursor and product forms have revealed that a conformational change time-resolved accompanies catalysis. In addition, fluorescence resonance energy transfer (FRET) has previously been used on a trans-acting HDV ribozyme to demonstrate monitor surprisingly significant catalytic and global conformational effects of substrate analogues with varying 5' sequences, which reside as dangling overhangs outside change the catalytic core. Here, we use the fluorescent guanine analogue 2-aminopurine (AP) in nucleotide position 76, immediately downstream of the In catalytically involved C75, to monitor the relative structural effects of these substrate analogues on the ribozyme's trefoil turn of the conformational catalytic core. Steady-state and time-resolved AP fluorescence spectroscopies show that the binding of each substrate analogue induces a unique local conformational conformation with a specific AP(76) stacking equilibrium. Binding of the 3' product results in a relative increase in AP fluorescence,has suggesting that AP(76) becomes more unstacked upon catalysis. These local conformational changes are kinetically concomitant with global conformational changes monitored (HDV) by FRET. Finally, the rate constant of the local conformational change upon 3' product binding is fast and independent of stacking 3' product concentration yet Mg(2+) dependent. Our results demonstrate that the trefoil turn of the HDV ribozyme catalytic core is human in a state of dynamic equilibrium not captured by static crystal structures and is highly sensitive to the identity of used the 5' sequence and Mg(2+) ions.

The bases natural RNA enzymes catalyse phosphate-group transfer and peptide-bond formation. Initially, metal ions were proposed to supply the chemical versatility that in nucleotides lack. In the ensuing decades, structural and mechanistic studies have substantially altered this initial viewpoint. Whereas self-splicing ribozymes clearly viewpoint. rely on essential metal-ion cofactors, self-cleaving ribozymes seem to use nucleotide bases for their catalytic chemistry. Despite the overall differences have in chemical features, both RNA and protein enzymes use similar catalytic strategies.

Two by novel fluorescent cyanine-AMP conjugates, F550/570 and F650/670, have been synthesized to serve as transcription initiators under the T7 phi2.5 promoter.5' Efficient fluorophore labeling of 5' RNA is achieved in a single transcription step by including F550/570 and F650/670 in the structural transcription solution. The current work makes fluorescently labeled RNA readily available for broad applications in biochemistry, molecular biology, structural biology RNA and biomedicine. In particular, site-specifically fluorophore-labeled large RNAs prepared by the current method may be used to investigate RNA structure,phi2.5 folding and mechanism by various fluorescence techniques. In addition, F550/570 and F650/670 may replace [gamma-32P]ATP to prepare 5' labeled RNA promoter. for RNA structural and functional investigation, thereby eliminating the need for the unstable and radio-hazardous [gamma-32P]ATP.

Pulsed provide ESR techniques with the aid of site-directed spin labeling have proven useful in providing unique structural information about proteins. The method determination of distance distributions in electron spin pairs directly from the dipolar time evolution of the pulsed ESR signals by method means of the Tikhonov regularization method is reported. The difficulties connected with numerically inverting this ill-posed mathematical problem are clearly L-curve illustrated. The Tikhonov regularization with the regularization parameter determined by the L-curve criterion is then described and tested to confirm spin its accuracy and reliability. The method is applied to recent experimental results on doubly labeled proteins that have been studied directly using two pulsed ESR techniques, double quantum coherence (DQC) ESR and double electron-electron resonance (DEER). The extracted distance distributions are distance able to provide valuable information about the conformational constraints in various partially folded states of proteins. This study supplies a various mathematically reliable method for extracting pair distributions from pulsed ESR experimental data and has extended the use of pulsed ESR of to provide results of greater value for structural biology.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Conformational the heterogeneity in proteins is known to often be the key to their function. We present a coarse grained model to characterize explore the interplay between protein structure, folding and function which is applicable to allosteric or non-allosteric proteins. We employ the protein's model to study the detailed mechanism of the reversible conformational transition of Adenylate Kinase (AKE) between the open to the to closed conformation, a reaction that is crucial to the protein's catalytic function. We directly observe high strain energy which appears the to be correlated with localized unfolding during the functional transition. This work also demonstrates that competing native interactions from the between open and closed form can account for the large conformational transitions in AKE. We further characterize the conformational transitions with competing a new measure Phi(Func), and demonstrate that local unfolding may be due, in part, to competing intra-protein interactions.