Structural and kinetic characterization of an acyl transferase ribozyme

We have previously isolated, by in vitro selection, an acyl-transferase ribozyme that is capable of transferring a biotinylated methionyl group from the 3' end of a hexanucleotide substrate to its own 5'-hydroxyl. Comparison of the sequences of a family of evolved derivatives of this ribozyme allowed us to generate a model of the secondary structure of the ribozyme. The predicted secondary structure was extensively tested and confirmed by single-mutant and compensatory double-mutant analyses. The role of the template domain in aligning the acyl-donor oligonucleotide and acyl-acceptor region of the ribozyme was confirmed in a similar manner. The significance of different domains of the ribozyme structure and the importance of two tandem G:U wobble base pairs in the template domain were studied by kinetic characterization of mutant ribozymes. The wobble base pairs contribute to the catalytic rate enhancement, but only in the context of the complete ribozyme; the ribozyme in turn alters the metal binding properties of this site. Competitive inhibition experiments with unacylated substrate oligonucleotide are consistent with the ribozyme acting to stabilize substrate binding to the template, while negative interactions with the aminoacyl portion of the substrate destabilize binding.     

Probing general base catalysis in the hammerhead ribozyme

Recent structural and computational studies have shed new light on the catalytic mechanism and active site structure of the RNA cleaving hammerhead ribozyme. Consequently, specific ribozyme functional groups have been hypothesized to be directly involved in general/acid base catalysis. In order to test this hypothesis, we have developed an affinity label to identify the functional general base in the S. mansoni hammerhead ribozyme. The ribozyme was reacted with a substrate analogue bearing a 2'-bromoacetamide group in place of the nucleophilic 2'-hydroxyl group which would normally be deprotonated by a general base. The electrophilic 2'-bromoacetamide group is poised to alkylate the general base, which is subsequently identified by footprinting analysis. Herein, we demonstrate alkylation of N1 of G12 in the hammerhead ribozyme in a pH and [Mg(2+)] dependent manner that is consistent with the native cleavage reaction. These results provide substantial evidence that deprotonated N1 of G12 functions directly as a general base in the hammerhead ribozyme; moreover, our experiments provide evidence that the pKa of G12 is perturbed downward in the context of the active site structure. We also observed other pH-independent alkylations, which do not appear to reflect the catalytic mechanism, but offer further insight into ribozyme conformation and structure.     

Reduction of an aldehyde by a NADH/Zn2+ -dependent redox active ribozyme

We report here the ability of an alcohol dehydrogenase (ADH) ribozyme to reduce a benzaldehyde. While the ribozyme was initially evolved in vitro based on the activity for the NAD+-dependent oxidation of the benzyl alcohol, we found that this ADH ribozyme is also capable of reducing the aldehyde in the presence of NADH and Zn2+. The rate acceleration gained by ribozyme catalysis was more than 6 orders of magnitude larger than the spontaneous reaction. Although the reversibility of phosphordiester and acyl transfer reactions catalyzed by ribozymes was known, that of other chemical reactions has not been well established. This study has demonstrated the reversibility of a hydride transfer chemistry catalyzed by the ADH ribozyme. Most interestingly, the ribozyme shares many features with the protein ADHs, e.g., reversibility and NADH/Zn2+ dependence.     

Diels-Alder ribozyme catalysis: a computational approach

A computational comparison of the Diels-Alder reaction of a maleimide and an anthracene in water and the active site of the ribozyme Diels-Alderase is reported. During the course of the catalyzed reaction, the maleimide is held in the hydrophobic pocket while the anthracene approaches to the maleimide through the back passage of the active site. The active site is so narrow that the anthracene has to adopt a tilted approach angle toward maleimide. The conformation of the active site changes marginally at different states of the reaction. Active site dynamics contribution to catalysis has been ruled out. The active site stabilizes the product more than the transition state (TS). The reaction coordinates of the ribozyme reaction in TS, RC1-CD1 and RC4-CD2, are 2.35 and 2.33 A, respectively, compared to 2.37 and 2.36 A in water. The approach angle of anthracene toward maleimide is twisted by 18 degrees in the TS structure of ribozyme reaction while no twisted angle is found in TS of the reaction in water. The free energy barriers for reactions in both ribozyme and water were obtained by umbrella sampling combined with SCCDFTB/MM. The calculated free energy barriers for the ribozyme and water reactions are in good agreement with the experimental values. As expected, Mulliken charges of the atoms involved in the ribozyme reaction change in a similar manner as that of the reaction in water. The proficiency of the Diels-Alder ribozyme reaction originates from the active site holding the two reactants in reactive conformations, in which the reacting atoms are brought together in van der Waals distances and reactants approach to each other at an appropriate angle.     

Probing the active site of a diels-alderase ribozyme by photoaffinity cross-linking

The active site of a Diels-Alderase ribozyme is located in solution by photoaffinity cross-linking using a productlike azidobenzyl probe. Two key nucleotides are identified that contact the Diels-Alder product in a conformation-dependent fashion. The design of such probes does not require knowledge of the three-dimensional structure of the ribozyme, and the technique yields both static and dynamic structural information. This work establishes photoaffinity cross-linking as an empirical approach that is applied here for the first time to an artificial ribozyme.     

RNA-catalyzed RNA ligation on an external RNA template

Variants of the hc ligase ribozyme, which catalyzes ligation of the 3' end of an RNA substrate to the 5' end of the ribozyme, were utilized to evolve a ribozyme that catalyzes ligation reactions on an external RNA template. The evolved ribozyme catalyzes the joining of an oligonucleotide 3'-hydroxyl to the 5'-triphosphate of an RNA hairpin molecule. The ribozyme can also utilize various substrate sequences, demonstrating a largely sequence-independent mechanism for substrate recognition. The ribozyme also carries out the ligation of two oligonucleotides that are bound at adjacent positions on a complementary template. Finally, it catalyzes addition of mononucleoside 5'-triphosphates onto the 3' end of an oligonucleotide primer in a template-dependent manner. The development of ribozymes that catalyze polymerase-type reactions contributes to the notion that an RNA world could have existed during the early history of life on Earth.     

The glmS ribozyme tunes the catalytically critical pK(a) of its coenzyme glucosamine-6-phosphate

The glmS ribozyme riboswitch is the first known natural catalytic RNA that employs a small-molecule cofactor. Binding of glucosamine-6-phosphate (GlcN6P) uncovers the latent self-cleavage activity of the RNA, which adopts a catalytically competent conformation that is nonetheless inactive in the absence of GlcN6P. Structural and analogue studies suggest that the amine of GlcN6P functions as a general acid-base catalyst, while its phosphate is important for binding affinity. However, the solution pK(a) of the amine is 8.06 ± 0.05, which is not optimal for proton transfer. Here we used Raman crystallography directly to determine the pK(a)'s of GlcN6P bound to the glmS ribozyme. Binding to the RNA lowers the pK(a) of the amine of GlcN6P to 7.26 ± 0.09 and raises the pK(a) of its phosphate to 6.35 ± 0.09. Remarkably, the pK(a)'s of these two functional groups are unchanged from their values for free GlcN6P (8.06 ± 0.05 and 5.98 ± 0.05, respectively) when GlcN6P binds to the catalytically inactive but structurally unperturbed G40A mutant of the ribozyme, thus implicating the ribozyme active site guanine in pK(a) tuning. This is the first demonstration that a ribozyme can tune the pK(a) of a small-molecule ligand. Moreover, the anionic glmS ribozyme in effect stabilizes the neutral amine of GlcN6P by lowering its pK(a). This is unprecedented and illustrates the chemical sophistication of ribozyme active sites.     

Fast cleavage kinetics of a natural hammerhead ribozyme

The hammerhead ribozyme is a small RNA motif that catalyzes the cleavage and ligation of RNA. The well-studied minimal hammerhead motif is inactive under physiological conditions and requires high Mg(2+) concentrations for efficient cleavage. In contrast, natural hammerheads are active under physiological conditions and contain motifs outside the catalytic core that lower the requirement for Mg(2+). Single-turnover kinetics were used here to characterize the Mg(2+) and pH dependence for cleavage of a trans-cleaving construct of the Schistosoma mansoni natural hammerhead ribozyme. Compared to the minimal hammerhead motif, the natural Schistosoma ribozyme requires 100-fold less Mg(2+) to achieve a cleavage rate of 1 min(-1). The improved catalysis results from tertiary interactions between loops in stems I and II and likely arises from increasing the population of the active conformation. Under optimum pH and Mg(2+) conditions this ribozyme cleaves at over 870 min(-1) at 25 degrees C, further demonstrating the impressive catalytic power of this ribozyme.     

Cross-catalytic replication of an RNA ligase ribozyme

A self-replicating RNA ligase ribozyme was converted to a cross-catalytic format whereby two ribozymes direct each other's synthesis from a total of four component substrates. Each ribozyme binds two RNA substrates and catalyzes their ligation to form the opposing ribozyme. The two ribozymes are not perfectly complementary, as is the case for replicating nucleic acid genomes in biology. Rather, the ribozymes contain both template elements, which are complementary, and catalytic elements, which are identical. The specificity of the template interactions allows the cross-catalytic pathway to dominate over all other reaction pathways. As the concentration of the two ribozymes increases, the rate of formation of additional ribozyme molecules increases, consistent with the overall autocatalytic behavior of the reaction system.     

Single-cell detection of trans-splicing ribozyme in vivo activity

The Tetrahymena trans-splicing ribozyme can edit RNA in a sequence-specific manner, but its efficiency needs to be improved for any functional rescues. This communication describes a simple method that uses a bacterial enzyme beta-lactamase to report trans-splicing activity of Tetrahymena ribozyme in single living mammalian cells by fluorescence microscopy and flow cytometry. This enzyme-based single-cell detection method is highly sensitive and compatible with living cell flow cytometry, and should allow a cell-based systematic screening of a vast library of ribozymes for better trans-spliced ribozyme variants.     

Direct measurement of a pK(a) near neutrality for the catalytic cytosine in the genomic HDV ribozyme using Raman crystallography

The hepatitis delta virus (HDV) ribozyme uses a cytosine to facilitate general acid-base catalysis. Biochemical studies suggest that C75 has a pKa perturbed to near neutrality. To measure this pKa directly, Raman spectra were recorded on single ribozyme crystals using a Raman microscope. A spectral feature arising from a single neutral cytosine was identified at 1528 cm(-1). At low pH, this mode was replaced with a new spectral feature. Monitoring these features as a function of pH revealed pKa values for the cytosine that couple anticooperatively with Mg2+ binding, with values of 6.15 and 6.40 in the presence of 20 and 2 mM Mg2+, respectively. These pKa values agree well with those obtained from ribozyme activity experiments in solution. To correlate the observed pKa with a specific nucleotide, crystals of C75U, which is catalytically inactive, were examined. The Raman difference spectra show that this mutation does not affect the conformation of the ribozyme. However, crystals of C75U did not produce a signal from a protonatable cytosine, providing strong evidence that protonation of C75 is being monitored in the wild-type ribozyme. These studies provide the first direct physical measurement of a pKa near neutrality for a catalytic residue in a ribozyme and show that ribozymes, like their protein enzyme counterparts, can optimize the pKa of their side chains for proton transfer.     

An unusual pH-independent and metal-ion-independent mechanism for hairpin ribozyme catalysis

Background: Hairpin ribozymes (RNA enzymes) catalyze the same chemical reaction as ribonuclease A and yet RNAs do not usually have functional groups analogous to the catalytically essential histidine and lysine sidechains of protein ribonucleases. Some RNA enzymes appear to recruit metal ions to act as Lewis acids in charge stabilization and metal-bound hydroxide for general base catalysis, but it has been reported that the hairpin ribozyme functions in the presence of metal ion chelators. This led us to investigate whether the hairpin ribozyme exploits a metal-ion-independent catalytic strategy.Results: Substitution of sulfur for nonbridging oxygens of the reactive phosphate of the hairpin ribozyme has small, stereospecific and metal-ionindependent effects on cleavage and ligation mediated by this ribozyme. Cobalt hexammine, an exchange-inert metal complex, supports full hairpin ribozyme activity, and the ribozyme's catalytic rate constants display only a shallow dependence on pH.Conclusions: Direct metal ion coordination to phosphate oxygens is not essential for hairpin ribozyme catalysis and metal-bound hydroxide does not serve as the general base in this catalysis. Several models might account for the unusual pH and metal ion independence: hairpin cleavage and ligation might be limited by a slow conformational change; a pH-independent or metalcation-independent chemical step, such as breaking the 5′ oxygen-phosphorus bond, might be rate determining; or finally, functional groups within the ribozyme might participate directly in catalytic chemistry. Whichever the case, the hairpin ribozyme appears to employ a unique strategy for RNA catalysis.     

Capreomycin and hygromycin B modulate the catalytic activity of the delta ribozyme in a manner that depends on the protonation and complexation with Cu2+ ions of these antibiotics

Catalytic RNA molecules (ribozymes) have often been used for the testing of interactions of antibiotics with ribonucleic acids. We showed that the impact of capreomycin and hygromycin B on delta ribozyme catalysis might change dramatically, from stimulation to inhibition, depending on conditions. In order to evaluate possible mechanisms of modulation of the ribozyme catalytic activity we used our earlier data on species distribution for protonated forms of capreomycin and hygromycin B and their complexes with Cu(2+) ions at different pH values. We proposed that, upon inhibition, the protonated amino group of capreomycin was located in the ribozyme catalytic cleft interfering with binding catalytic Mg(2+). Such a mechanism was also supported by the results of ribozyme inhibition with capreomycin complexed with Cu(2+). The effects of stimulation of the delta ribozyme activity by capreomycin and hygromycin B were less pronounced than inhibition. Possibly, the amino functions of these antibiotics might be involved in a general acid-base catalysis performed by the ribozyme, acting as proton acceptors/donors.     

Relationship between catalytic activity and secondary structure of a hammerhead ribozyme: A study using thermodynamic parameters for RNA structure prediction

Abstract

The stabilization energy for the secondary structures of wild-type hammerhead and mutant ribozymes has been calculated at different salt conditions and temperatures by using the thermodynamic parameters for RNA structure prediction. The most stable structure at each condition has been searched and the obtained secondary structure is compared with the structure suggested phylogenetically or experimentally. The results indicate that the hammerhead-type secondary structure of the ribozyme and its reactivity correlate with each other. The multibranched loop containing the self-cleavage site of the ribozyme particularly should be a key structure in the hammerhead ribozyme reaction. The predicted secondary structures also suggest that the reactivity of the hammerhead ribozyme should be very much lower at 10°C than that at 37°C.     

High‐Throughput Mutational Analysis of a Twister Ribozyme

Recent discoveries of new classes of self‐cleaving ribozymes in diverse organisms have triggered renewed interest in the chemistry and biology of ribozymes. Functional analysis and engineering of ribozymes often involve performing biochemical assays on multiple ribozyme mutants. However, because each ribozyme mutant must be individually prepared and assayed, the number and variety of mutants that can be studied are severely limited. All of the single and double mutants of a twister ribozyme (a total of 10 296 mutants) were generated and assayed for their self‐cleaving activity by exploiting deep sequencing to count the numbers of cleaved and uncleaved sequences for every mutant. Interestingly, we found that the ribozyme is highly robust against mutations such that 71 % and 30 % of all single and double mutants, respectively, retain detectable activity under the assay conditions. It was also observed that the structural elements that comprise the ribozyme exhibit distinct sensitivity to mutations.     

A versatile tRNA aminoacylation catalyst based on RNA

Aminoacyl-tRNA synthetase (ARS) ribozymes have potential to develop a novel genetic coding system. Although we have previously isolated such a ribozyme that recognizes aromatic amino acids, it could not be used as a versatile catalyst due to its limited ability of aminoacylation to a particular tRNA used for the selection. To overcome this limitation, we used a combination of evolutionary and engineering approaches to generate an optimized ribozyme. The ribozyme, consisting of 45 nucleotides, displays a broad spectrum of activity toward various tRNAs. Most significantly, this ribozyme is able to exhibit multiple turnover activity and charge parasubstituted Phe analogs onto an engineered suppressor tRNA (tRNA(Asn)(CCCG)). Thus, it provides a useful and flexible tool for the custom synthesis of mischarged tRNAs with natural and nonnatural amino acids.     

Active site labeling of G8 in the hairpin ribozyme: implications for structure and mechanism

There is mounting evidence that suggests that general acid/base catalysis is operative in the hairpin ribozyme, with analogy to the protein enzyme RNaseA. Nevertheless, the extent of general base catalysis as well as the identity of the specific chemical groups responsible remains the subject of some controversy. An affinity label has previously been used to alkylate histidine 12 (His12), the active general base in RNaseA. To date, no such experiment has been applied to a ribozyme. We have synthesized the analogous affinity label for the hairpin ribozyme with an electrophilic 2'-bromoacetamide group in lieu of the 2'-hydroxyl (2'OH) at the substrate cleavage site and show that guanosine 8 (G8) of the hairpin ribozyme is specifically alkylated, most likely at the N1 position. This evidence strongly implicates N1 of G8 in active site chemistry. By direct analogy to RNase A, these findings could be consistent with the hypothesis that deprotonated G8 residue functions as a general base in the hairpin ribozyme. Other mechanistic possibilities for N1 of G8 such as indirect general base catalysis mediated by a water molecule or transition state stabilization could also be consistent with our findings.     

Novel RNA catalysts for the Michael reaction

BACKGROUND: In vitro selected ribozymes with nucleotide synthase, peptide and carbon-carbon bond forming activity provide insight into possible scenarios on how chemical transformations may have been catalyzed before protein enzymes had evolved. Metabolic pathways based on ribozymes may have existed at an early stage of evolution. RESULTS: We have isolated a novel ribozyme that mediates Michael-adduct formation at a Michael-acceptor substrate, similar to the rate-limiting step of the mechanistic sequence of thymidylate synthase. The kinetic characterization of this catalyst revealed a rate enhancement by a factor of approximately 10(5). The ribozyme shows substrate specificity and can act as an intermolecular catalyst which transfers the Michael-donor substrate onto an external 20-mer RNA oligonucleotide containing the Michael-acceptor system. CONCLUSION: The ribozyme described here is the first example of a catalytic RNA with Michael-adduct forming activity which represents a key mechanistic step in metabolic pathways and other biochemical reactions. Therefore, previously unforeseen RNA-evolution pathways can be considered, for example the formation of dTMP from dUMP. The substrate specificity of this ribozyme may also render it useful in organic syntheses.     

Real-Time Characterization of Ribozymes by Fluorecence Resonance Energy Transfer (FRET)

More rapid than with conventional methods is the analysis of ribozyme kinetics upon use of FRET substrates. In these substrates a fluorophore and a fluorescence-quenching molecule lie in close spatial proximity. The intramolecular fluorescence quenching is neutralized upon cleavage, and the fluorescence intensity is a measure of the ribozyme activity. By automation and computer assistance the activity of ribozymes can be monitored under high-throughput conditions.     

Probing the binding of Tb(III) and Eu(III) to the hammerhead ribozyme using luminescence spectroscopy

BACKGROUND: Divalent metal ions serve as structural as well as catalytic cofactors in the hammerhead ribozyme reaction. The natural cofactor in these reactions is Mg(II), but its spectroscopic silence makes it difficult to study. We previously showed that a single Tb(III) ion inhibits the hammerhead ribozyme by site-specific competition for a Mg(II) ion and therefore can be used as a spectroscopic probe for the Mg(II) it replaces. RESULTS: Lanthanide luminescence spectroscopy was used to study the coordination environment around Tb(III) and Eu(III) ions bound to the structurally well-characterized site on the hammerhead ribozyme. Sensitized emission and direct excitation experiments show that a single lanthanide ion binds to the ribozyme under these conditions and that three waters of hydration are displaced from the Tb(III) upon binding the RNA. Furthermore, we show that these techniques allow the comparison of binding affinities for a series of ions to this site. The binding affinities for ions at the G5 site correlates linearly with the function Z(2)/r of the aqua ion (where Z is the charge and r is the radius of the ion). CONCLUSIONS: This study compares the crystallographic nature of the G5 metal-binding site with solution measurements and gives a clearer picture of the coordination environment of this ion. These results provide one of the best characterized metal-binding sites from a ribozyme, so we use this information to compare the RNA site with that of typical metalloproteins.