Capturing hammerhead ribozyme structures in action by modulating general base catalysis

We have obtained precatalytic (enzyme–substrate complex) and postcatalytic (enzyme–product complex) crystal structures of an active full-length hammerhead RNA that cleaves in the crystal. Using the natural satellite tobacco ringspot virus hammerhead RNA sequence, the self-cleavage reaction was modulated by substituting the general base of the ribozyme, G12, with A12, a purine variant with a much lower pK_a that does not significantly perturb the ribozyme's atomic structure. The active, but slowly cleaving, ribozyme thus permitted isolation of enzyme–substrate and enzyme–product complexes without modifying the nucleophile or leaving group of the cleavage reaction, nor any other aspect of the substrate. The predissociation enzyme-product complex structure reveals RNA and metal ion interactions potentially relevant to transition-state stabilization that are absent in precatalytic structures.     

Tertiary contacts distant from the active site prime a ribozyme for catalysis

Minimal hammerhead ribozymes have been characterized extensively by static and time-resolved crystallography as well as numerous biochemical analyses, leading to mutually contradictory mechanistic explanations for catalysis. We present the 2.2 A resolution crystal structure of a full-length Schistosoma mansoni hammerhead ribozyme that permits us to explain the structural basis for its 1000-fold catalytic enhancement. The full-length hammerhead structure reveals how tertiary interactions occurring remotely from the active site prime this ribozyme for catalysis. G-12 and G-8 are positioned consistent with their previously suggested roles in acid-base catalysis, the nucleophile is aligned with a scissile phosphate positioned proximal to the A-9 phosphate, and previously unexplained roles of other conserved nucleotides become apparent within the context of a distinctly new fold that nonetheless accommodates the previous structural studies. These interactions permit us to explain the previously irreconcilable sets of experimental results in a unified, consistent, and unambiguous manner.     

Structure—function studies of the hammerhead ribozyme

Elucidation of the catalytic mechanism and structure—function relationship studies of the hammerhead ribozyme continue to be an area of intensive research. A combination of diverse approaches, such as X ray crystallography, spectral studies, chemical modifications, sequence variations and kinetic analyses, have provided valuable insight into the cleavage mechanism of this ribozyme. The hammerhead ribozyme crystal structures have provided valuable insight into conformational deformations needed to attain the catalytically active structure. Similarly, determination of ribozyme solution structure by spectroscopic analyses and the effect of divalent metal ions on RNA folding has further aided in the construction of a model for hammerhmead catalysis.     

A pH-dependent conformational change, rather than the chemical step, appears to be rate-limiting in the hammerhead ribozyme cleavage reaction.

We have investigated the chemical basis for a previously observed 7.8 A conformational change in the hammerhead ribozyme that positions the substrate for in-line attack. We have found that the conformational change can only be observed at or above pH 8.5 (in the presence of Co(2+)) and requires the presence of an ionizable 2'-OH at the cleavage site, and note that this observed apparent pK(a) of 8.5 for the conformational change is within experimental error (+/-0.5) of the previously reported apparent kinetic pK(a) of 8.5 for the hammerhead ribozyme in the presence of Co(2+). We have solved two crystal structures of hammerhead ribozymes having 2'-OCH(3) or 2'-F substitutions at the cleavage site and have found that these will not undergo a conformational change equivalent to that observed for the hammerhead ribozyme having an unmodified attacking nucleophile under otherwise identical conditions. We have also characterized the kinetics of cleavage in the crystal. In addition to verifying that the particular sequence of RNA that we crystallized cleaves faster in the crystal than in solution, we also find that the extent of cleavage in the crystal is complete, unlike in solution where this and most other hammerhead ribozyme substrates are cleaved only to about 70 % completion. The initial cleavage rate in the crystal obeys the expected log-linear relation between cleavage-rate and pH with a slope of 0.7, as has been observed for other hammerhead ribozyme sequences in solution, indicating that in both the crystal and in solution the pH-dependent step is rate-limiting. However, the cleavage rate in the crystal is biphasic, with the most dramatic distinction between initial (slower) and final (faster) phases appearing at pH 6.0. The initial phase corresponds to the pH-dependent cleavage rate observed in solution, but the second, faster phase is roughly pH-independent and closely parallels the cleavage rate observed at pH 8 (0.4/minute). This result is particularly remarkable because it entails that the rapidly cleaving phase at pH 6 is comparable to the cleavage rate for the fastest cleaving hammerhead ribozymes at pH 6. Based upon these observations, we conclude that the pH-dependent conformational change is the rate-determining step under standard conditions for the hammerhead ribozyme self-cleavage reaction, and that an ionizable 2'-proton at cleavage site is required for this conformational change. We further hypothesize that deprotonation of the cleavage-site 2'-oxygen drives this conformational change.     

Molecular dynamics investigations of hammerhead ribozyme RNA

The hammerhead ribozyme, a small catalytic RNA molecule, cleaves, in the presence of magnesium ions, a specific phosphodiester bond within its own backbone, leading to 23-cyclic phosphate and 5-OH extremities. In order to study the dynamical flexibility of the hammerhead RNA, we performed molecular dynamics simulations of the solvated crystal structure of an active hammerhead ribozyme, obtained after flash-freezing crystals soaked with magnesium. Because of a careful equilibration protocol and the use of the Ewald summation in calculating the electrostatic interactions, the RNA structure remained close to the crystal structure, as attested by a root-mean-square deviation below 2.5 A after 750 ps of simulation. All Watson-Crick base pairs were intact at the end of the simulations. The tertiary interactions, such as the sheared G.A pairs and the U-turn, important for the stabilisation of the three-dimensional RNA fold, were also retained. The results demonstrate that molecular dynamics simulations can be successfully used to investigate the dynamical behaviour of a ribozyme, thus, opening a road to study the role of transient structural changes involved in ribozyme catalysis.     

A helical twist-induced conformational switch activates cleavage in the hammerhead ribozyme

We have captured the structure of a pre-catalytic conformational intermediate of the hammerhead ribozyme using a phosphodiester tether formed between I and Stem II. This phosphodiester tether appears to mimic interactions in the wild-type hammerhead RNA that enable switching between nuclease and ligase activities, both of which are required in the replicative cycles of the satellite RNA viruses from which the hammerhead ribozyme is derived. The structure of this conformational intermediate reveals how the attacking nucleophile is positioned prior to cleavage, and demonstrates how restricting the ability of Stem I to rotate about its helical axis, via interactions with Stem II, can inhibit cleavage. Analogous covalent crosslinking experiments have demonstrated that imposing such restrictions on interhelical movement can change the hammerhead ribozyme from a nuclease to a ligase. Taken together, these results permit us to suggest that switching between ligase and nuclease activity is determined by the helical orientation of Stem I relative to Stem II.     

Existence of efficient divalent metal ion-catalyzed and inefficient divalent metal ion-independent channels in reactions catalyzed by a hammerhead ribozyme

The hammerhead ribozyme is generally accepted as a well characterized metalloenzyme. However, the precise nature of the interactions of the RNA with metal ions remains to be fully defined. Examination of metal ion-catalyzed hammerhead reactions at limited concentrations of metal ions is useful for evaluation of the role of metal ions, as demonstrated in this study. At concentrations of Mn²⁺ ions from 0.3 to 3 mM, addition of the ribozyme to the reaction mixture under single-turnover conditions enhances the reaction with the product reaching a fixed maximum level. Further addition of the ribozyme inhibits the reaction, demonstrating that a certain number of divalent metal ions is required for proper folding and also for catalysis. At extremely high concentrations, monovalent ions, such as Na⁺ ions, can also serve as cofactors in hammerhead ribozyme-catalyzed reactions. However, the catalytic efficiency of monovalent ions is extremely low and, thus, high concentrations are required. Furthermore, addition of monovalent ions to divalent metal ion-catalyzed hammerhead reactions inhibits the divalent metal ion-catalyzed reactions, suggesting that the more desirable divalent metal ion–ribozyme complexes are converted to less desirable monovalent metal ion–ribozyme complexes via removal of divalent metal ions, which serve as a structural support in the ribozyme complex. Even though two channels appear to exist, namely an efficient divalent metal ion-catalyzed channel and an inefficient monovalent metal ion-catalyzed channel, it is clear that, under physiological conditions, hammerhead ribozymes are metalloenzymes that act via the significantly more efficient divalent metal ion-dependent channel. Moreover, the observed kinetic data are consistent with Lilley’s and DeRose’s two-phase folding model that was based on ground state structure analyses.     

Ion-induced folding of the hammerhead ribozyme: a fluorescence resonance energy transfer study.

The ion-induced folding transitions of the hammerhead ribozyme have been analysed by fluorescence resonance energy transfer. The hammerhead ribozyme may be regarded as a special example of a three-way RNA junction, the global structure of which has been studied by comparing the distances (as energy transfer efficiencies) between the ends of pairs of labelled arms for the three possible end-to-end vectors as a function of magnesium ion concentration. The data support two sequential ion-dependent transitions, which can be interpreted in the light of the crystal structures of the hammerhead ribozyme. The first transition corresponds to the formation of a coaxial stacking between helices II and III; the data can be fully explained by a model in which the transition is induced by a single magnesium ion which binds with an apparent association constant of 8000-10 000 M-1. The second structural transition corresponds to the formation of the catalytic domain of the ribozyme, induced by a single magnesium ion with an apparent association constant of approximately 1100 M-1. The hammerhead ribozyme provides a well-defined example of ion-dependent folding in RNA.     

An ultraviolet crosslink in the hammerhead ribozyme dependent on 2-thiocytidine or 4-thiouridine substitution.

The hammerhead domain is one of the smallest known ribozymes. Like other ribozymes it catalyzes site-specific cleavage of a phosphodiester bond. The hammerhead ribozyme has been the subject of a vast number of biochemical and structural studies aimed at determining the structure and mechanism of cleavage. Recently crystallographic analysis has produced a structure for the hammerhead. As the hammerhead is capable of undergoing cleavage within the crystal, it would appear that the crystal structure is representative of the catalytically active solution structure. However, the crystal structure conflicts with much of the biochemical data and reveals a catalytic metal ion binding site expected to be of very low affinity. Clearly, additional studies are needed to reconcile the discrepancies and provide a clear understanding of the structure and mechanism of the hammerhead ribozyme. Here we demonstrate that a unique crosslink can be induced in the hammerhead with 2-thiocytidine or 4-thiouridine substitution at different locations within the conserved core. Generation of the same crosslink with different modifications at different positions suggests that the structure trapped by the crosslink may be relevant to the catalytically active solution structure of the hammerhead ribozyme. As this crosslink appears to be incompatible with the crystal structure, this provides yet another indication that the active solution and crystal structures may differ significantly.     

Pseudoknot Interaction-Mediated Activation of Type I Hammerhead Ribozyme: A New Class of Gene-Therapeutic Agents

Recently discovered hammerhead ribozymes that are activated through pseudoknot interactions (Watson-Crick base pairs between loops) are attractive candidates as gene-therapeutic agents because sequences of gene-therapeutic ribozymes can be designed simply based on the sequence complementarity against target RNAs. Herein, we examined if the newly found pseudoknot-type hammerhead ribozyme with type I topology is activated through the pseudoknot interactions. Substitutions of pseudoknot sequences into fully mismatched ones significantly reduced the activity of type I pseudoknot-type hammerhead ribozyme, while those with full-matched pseudoknot sequences were highly active. The results indicated that the pseudoknot interactions activated type I pseudoknot-type hammerhead ribozyme, making them suitable as gene-therapeutic agents.     

Sequence requirements of the hammerhead RNA self-cleavage reaction.

A previously well-characterized hammerhead catalytic RNA consisting of a 24-nucleotide substrate and a 19-nucleotide ribozyme was used to perform an extensive mutagenesis study. The cleavage rates of 21 different substrate mutations and 24 different ribozyme mutations were determined. Only one of the three phylogenetically conserved base pairs but all nine of the conserved single-stranded residues in the central core are needed for self cleavage. In most cases the mutations did not alter the ability of the hammerhead to assemble into a bimolecular complex. In the few cases where mutant hammerheads did not assemble, it appeared to be the result of the mutation stabilizing an alternate substrate or ribozyme secondary structure. All combinations of mutant substrate and mutant ribozyme were less active than the corresponding single mutations, suggesting that the hammerhead contains few, if any, replaceable tertiary interactions as are found in tRNA. The refined consensus hammerhead resulting from this work was used to identify potential hammerheads present in a variety of Escherichia coli gene sequences.     

Interaction between hammerhead ribozyme and RNA substrates measured by a surface plasmon resonance biosensor

Dynamic interactions between hammerhead ribozymes and RNA substrates were measured using the surface plasmon resonance (SPR) technology. Two in vitro transcribed substrates (non-cleavable and cleavable) were immobilised on streptavidin-coated dextran matrices and subsequently challenged with non-related yeast tRNA or two hammerhead ribozymes, both of which had previously been shown to exhibit functional binding and cleavage of complementary target RNAs. The target-binding domain of one of the ribozymes was fully complementary to a 16-ribonucleotide stretch on the immobilised substrates, while the other ribozyme had a nine-ribonucleotide complementarity. The two ribozymes could readily be differentiated with regard to affinity. Cleavage could be measured, using the ribozyme with full target complementarity to the cleavable substrate. In contrast, the ribozyme with lower affinity lacked cleavage activity. We suggest that SPR will be useful for investigations of ribozyme-substrate interactions.     

The tolerance to exchanges of the Watson Crick base pair in the hammerhead ribozyme core is determined by surrounding elements

Tertiary interacting elements are important features of functional RNA molecules, for example, in all small nucleolytic ribozymes. The recent crystal structure of a tertiary stabilized type I hammerhead ribozyme revealed a conventional Watson-Crick base pair in the catalytic core, formed between nucleotides C3 and G8. We show that any Watson-Crick base pair between these positions retains cleavage competence in two type III ribozymes. In the Arabidopsis thaliana sequence, only moderate differences in cleavage rates are observed for the different base pairs, while the peach latent mosaic viroid (PLMVd) ribozyme exhibits a preference for a pyrimidine at position 3 and a purine at position 8. To understand these differences, we created a series of chimeric ribozymes in which we swapped sequence elements that surround the catalytic core. The kinetic characterization of the resulting ribozymes revealed that the tertiary interacting loop sequences of the PLMVd ribozyme are sufficient to induce the preference for Y3-R8 base pairs in the A. thaliana hammerhead ribozyme. In contrast to this, only when the entire stem-loops I and II of the A. thaliana sequences are grafted on the PLMVd ribozyme is any Watson-Crick base pair similarly tolerated. The data provide evidence for a complex interplay of secondary and tertiary structure elements that lead, mediated by long-range effects, to an individual modulation of the local structure in the catalytic core of different hammerhead ribozymes.     

Nucleic acid binding properties of the nucleic acid chaperone domain of hepatitis delta antigen

The N terminal region of hepatitis delta antigen (HDAg), referred to here as NdAg, has a nucleic acid chaperone activity that modulates the ribozyme activity of hepatitis delta virus (HDV) RNA and stimulates hammerhead ribozyme catalysis. We characterized the nucleic acid binding properties of NdAg, identified the structural and sequence domains important for nucleic acid binding, and studied the correlation between the nucleic acid binding ability and the nucleic acid chaperone activity. NdAg does not recognize the catalytic core of HDV ribozyme specifically. Instead, NdAg interacts with a variety of nucleic acids and has higher affinities to longer nucleic acids. The studies with RNA homopolymers reveal that the binding site size of NdAg is around nine nucleotides long. The extreme N terminal portion of NdAg, the following coiled-coil domain and the basic amino acid clusters in these regions are important for nucleic acid binding. The nucleic acid–NdAg complex is stabilized largely by electrostatic interactions. The formation of RNA–protein complex appears to be a prerequisite for facilitating hammerhead ribozyme catalysis of NdAg and its derivatives. Mutations that reduce the RNA binding activity or high ionic strength that destabilizes the RNA–protein complex, reduce the nucleic acid chaperone activity of NdAg.     

Photocrosslinking detects a compact, active structure of the hammerhead ribozyme

The hammerhead ribozyme has been intensively studied for approximately 15 years, but its cleavage mechanism is not yet understood. Crystal structures reveal a Y-shaped molecule in which the cleavage site is not ideally aligned for an S(N)2 reaction and no RNA functional groups are positioned appropriately to perform the roles of acid and base or other functions in the catalysis. If the ribozyme folds to a more compact structure in the transition state, it probably does so only transiently. We have used photocrosslinking as a tool to trap hammerhead ribozyme-substrate complexes in various stages of folding. Results suggest that the two substrate residues flanking the cleavage site approach and stack upon two guanosines (G8 and G12) in domain 2, moving 10-15 A closer to domain 2 than they appear in the crystal structure. Most crosslinks obtained with the nucleotide analogues positioned in the ribozyme core are catalytically inactive; however, one cobalt(III) hexaammine-dependent crosslink of an unmodified ribozyme retains catalytic activity and confirms the close stacking of cleavage site residue C17 with nucleotide G8 in domain 2. These findings suggest that residues involved in the chemistry of hammerhead catalysis are likely located in that region containing G8 and G12.     

Zinc-dependent cleavage in the catalytic core of the hammerhead ribozyme: evidence for a pH-dependent conformational change

We have characterized a novel Zn²⁺-catalyzed cleavage site between nucleotides C3 and U4 in the catalytic core of the hammerhead ribozyme. In contrast to previously described divalent metal-ion-dependent cleavage of RNA, U4 cleavage is only observed in the presence of Zn²⁺. This new cleavage site has an unusual pH dependence, in that U4 cleavage products are only observed above pH 7.9 and reach a maximum yield at about pH 8.5. These data, together with the fact that no metal ion-binding site is observed in proximity to the U4 cleavage site in either of the crystal structures, point toward a pH-dependent conformational change in the hammerhead ribozyme. We have described previously Zn²⁺-dependent cleavage between G8 and A9 in the hammerhead ribozyme and have discovered that U4 cleavage occurs only after A9 cleavage. To our knowledge, this is the first example of sequential cleavage events as a possible regulatory mechanism in ribozymes.     

Folding of the hammerhead ribozyme: pyrrolo-cytosine fluorescence separates core folding from global folding and reveals a pH-dependent conformational change

The catalytic activity of the hammerhead ribozyme is limited by its ability to fold into the native tertiary structure. Analysis of folding has been hampered by a lack of assays that can independently monitor the environment of nucleobases throughout the ribozyme-substrate complex in real time. Here, we report the development and application of a new folding assay in which we use pyrrolo-cytosine (pyC) fluorescence to (1) probe active-site formation, (2) examine the ability of peripheral ribozyme domains to support native folding, (3) identify a pH-dependent conformational change within the ribozyme, and (4) explore its influence on the equilibrium between the folded and unfolded core of the hammerhead ribozyme. We conclude that the natural ribozyme folds in two distinct noncooperative steps and the pH-dependent correlation between core folding and activity is linked to formation of the G8-C3 base pair.     

The ubiquitous hammerhead ribozyme

The hammerhead ribozyme is a small catalytic RNA motif capable of endonucleolytic (self-) cleavage. It is composed of a catalytic core of conserved nucleotides flanked by three helices, two of which form essential tertiary interactions for fast self-scission under physiological conditions. Originally discovered in subviral plant pathogens, its presence in several eukaryotic genomes has been reported since. More recently, this catalytic RNA motif has been shown to reside in a large number of genomes. We review the different approaches in discovering these new hammerhead ribozyme sequences and discuss possible biological functions of the genomic motifs.     

An RNA chaperone activity of non-specific RNA binding proteins in hammerhead ribozyme catalysis.

We have previously shown that a protein derived from the p7 nucleocapsid (NC) protein of HIV type-1 increases kcat/Km and kcat for cleavage of a cognate substrate by a hammerhead ribozyme. Here we show directly that the increase in kcat/Km arises from catalysis of the annealing of the RNA substrate to the ribozyme and the increase in kcat arises from catalysis of dissociation of the RNA products from the ribozyme. A peptide polymer derived from the consensus sequence of the C-terminal domain of the hnRNP A1 protein (A1 CTD) provides similar enhancements. Although these effects apparently arise from non-specific interactions, not all non-specific binding interactions led to these enhancements. NC and A1 CTD exert their effects by accelerating attainment of the thermodynamically most stable species throughout the ribozyme catalytic cycle. In addition, NC protein is shown to resolve a misfolded ribozyme-RNA complex that is otherwise long lived. These in vitro results suggest that non-specific RNA binding proteins such as NC and hnRNP proteins may have a biological role as RNA chaperones that prevent misfolding of RNAs and resolve RNAs that have misfolded, thereby ensuring that RNA is accessible for its biological functions.