Satellite cereal yellow dwarf virus-RPV (satRPV) RNA requires a douXble hammerhead for self-cleavage and an alternative structure for replication.

The 110 nt hammerhead ribozyme in the satellite RNA of cereal yellow dwarf virus-RPV (satRPV RNA) folds into an alternative conformation that inhibits self-cleavage. This alternative structure comprises a pseudoknot with base-pairing between loop (L1) and a single-stranded bulge (L2a), which are located in hammerhead stems I and II, respectively. Mutations that disrupt this base-pairing, or otherwise cause the ribozyme to more closely resemble a canonical hammerhead, greatly increase self-cleavage. In a more natural multimeric sequence context containing the full-length satRPV RNA and two copies of the hammerhead, wild-type RNA cleaves much more efficiently than in the 110 nt context. Mutations in the upstream hammerhead, including a knock-out in the catalytic core, affect cleavage at the downstream cleavage site, indicating that multimers of satRPV RNA cleave via a double hammerhead. The double hammerhead includes base-pairing between two copies of the L1 sequence which extends stem I. Disruption of L1-L1 base-pairing slows cleavage of the multimer. L1-L2a base-pairing is required for efficient replication of satRPV RNA in oat protoplasts. Mutations that affect self-cleavage of the multimer do not correlate with replication efficiency, indicating that the ability to self-cleave is not a primary determinant of replication. We present a replication model in which multimeric satRPV RNA folds into alternative conformations that cannot form in the monomer. One potential metastable intermediate conformation involves L1-L2a base-pairing that may facilitate formation of the double hammerhead. However, we conclude that L1-L2a also performs some other essential function in the satRPV RNA replication cycle, because the L1-L2a base-pairing is more important than efficient self-cleavage for replication.     

Mutagenesis analysis of a self-cleaving RNA.

The hammerhead structural model proposed for sequences that mediate self-cleavage of certain RNAs contains base-paired three stems and 13 conserved bases. Insertion, deletion and base substitution mutations were carried out on a 58 base RNA containing the sequence of the single-hammerhead structure of the plus RNA of the virusoid of lucerne transient streak virus, and the effects on self-cleavage assessed. Results showed that there is flexibility in the sequence requirements for self-cleavage in vitro, but alterations of the conserved sequence or predicted secondary structure generally reduced the efficiency of self-cleavage.     

Probing the hammerhead ribozyme structure with ribonucleases.

Susceptibility to RNase digestion has been used to probe the conformation of the hammerhead ribozyme structure prepared from chemically synthesised RNAs. Less than about 1.5% of the total sample was digested to obtain a profile of RNase digestion sites. The observed digestion profiles confirmed the predicted base-paired secondary structure for the hammerhead. Digestion profiles of both cis and trans hammerhead structures were nearly identical which indicated that the structural interactions leading to self-cleavage were similar for both systems. Furthermore, the presence or absence of Mg2+ did not affect the RNase digestion profiles, thus indicating that Mg2+ did not modify the hammerhead structure significantly to induce self-cleavage. The base-paired stems I and II in the hammerhead structure were stable whereas stem III, which was susceptible to digestion, appeared to be an unstable region. The single strand domains separating the stems were susceptible to digestion with the exception of sites adjacent to guanosines; GL2.1 in the stem II loop and G12 in the conserved GAAAC sequence, which separates stems II and III. The absence of digestion at GL2.1 in the stem II hairpin loop of the hammerhead complex was maintained in uncomplexed ribozyme and in short oligonucleotides containing only the stem II hairpin region. In contrast, the G12 site became susceptible when the ribozyme was not complexed with its substrate. Overall the results are consistent with the role of Mg2+ in the hammerhead self-cleavage reaction being catalytic and not structural.     

Characterization of a native hammerhead ribozyme derived from schistosomes

A recent re-examination of the role of the helices surrounding the conserved core of the hammerhead ribozyme has identified putative loop-loop interactions between stems I and II in native hammerhead sequences. These extended hammerhead sequences are more active at low concentrations of divalent cations than are minimal hammerheads. The loop-loop interactions are proposed to stabilize a more active conformation of the conserved core. Here, a kinetic and thermodynamic characterization of an extended hammerhead sequence derived from Schistosoma mansoni is performed. Biphasic kinetics are observed, suggesting the presence of at least two conformers, one cleaving with a fast rate and the other with a slow rate. Replacing loop II with a poly(U) sequence designed to eliminate the interaction between the two loops results in greatly diminished activity, suggesting that the loop-loop interactions do aid in forming a more active conformation. Previous studies with minimal hammerheads have shown deleterious effects of Rp-phosphorothioate substitutions at the cleavage site and 5' to A9, both of which could be rescued with Cd2+. Here, phosphorothioate modifications at the cleavage site and 5' to A9 were made in the schistosome-derived sequence. In Mg2+, both phosphorothioate substitutions decreased the overall fraction cleaved without significantly affecting the observed rate of cleavage. The addition of Cd2+ rescued cleavage in both cases, suggesting that these are still putative metal binding sites in this native sequence.     

Mutagenesis analysis of the self-cleavage domain of hepatitis delta virus antigenomic RNA.

To determine the sequence requirements and structural features of the self-cleavage domain of hepatitis delta virus (HDV) antigenomic RNA, we constructed a series of mutants and measured the rate constant of the cleavage reaction for each. The self-cleavage activity of HDV RNA of antigenomic sense was found to reside in a region of less than 90 nucleotides in length. The catalytic domain contained a long complementary sequence which could be deleted to half of its original size. Moreover, this region could be replaced by other sequences as long as they could fold into a stem-and-loop structure. The catalytic domain also required a 6-basepair helix adjacent to the cleaving point for activity. The structural features of these two base-pairing regions are quite similar to those of the HDV genomic self-cleavage domain. The cleavage site as well as the the hinge region (the sequence between the two stems) requires specific sequences for activity.     

The ion-induced folding of the hammerhead ribozyme: core sequence changes that perturb folding into the active conformation.

The hammerhead ribozyme undergoes an ion-dependent folding process into the active conformation. We find that the folding can be blocked at specific stages by changes of sequence or functionality within the core. In the the absence of added metal ions, the global structure of the hammerhead is extended, with a large angle subtended between stems I and II. No core sequence changes appear to alter this geometry, consistent with an unstructured core under these conditions. Upon addition of low concentrations of magnesium ions, the hammerhead folds by an association of stems II and III, to include a large angle between them. This stage is inhibited or altered by mutations within the oligopurine sequence lying between stems II and III, and folding is completely prevented by an A14G mutation. Further increase in magnesium ion concentration brings about a second stage of folding in the natural sequence hammerhead, involving a reorientation of stem I, which rotates around into the same direction of stem II. Because this transition occurs over the same range of magnesium ion concentration over which the hammerhead ribozyme becomes active, it is likely that the final conformation is most closely related to the active form of the structure. Magnesium ion-dependent folding into this conformation is prevented by changes at G5, notably removal of the 2'-hydroxyl group and replacement of the base by cytidine. The ability to dissect the folding process by means of sequence changes suggests that two separate ion-dependent stages are involved in the folding of the hammerhead ribozyme into the active conformation.     

Thiophosphate interference experiments locate phosphates important for the hammerhead RNA self-cleavage reaction.

A hammerhead domain of less than 50 nucleotides is responsible for a self-cleavage reaction in the replication of plant RNA pathogens. The hammerhead is composed of three helices joining at a central conserved core of 11 single stranded nucleotides. The core is believed to fold into a tertiary structure that provides functional groups for catalysis and to coordinate one or more divalent metal ions. In this study we use a phosphorothioate substitution interference assay to identify four phosphates in the conserved core which also play a role in the self-cleavage reaction.     

Alternative tertiary structure attenuates self-cleavage of the ribozyme in the satellite RNA of barley yellow dwarf virus.

A self-cleaving satellite RNA associated with barley yellow dwarf virus (sBYDV) contains a sequence predicted to form a secondary structure similar to catalytic RNA molecules (ribozymes) of the 'hammerhead' class (Miller et al., 1991, Virology 183, 711-720). However, this RNA differs from other naturally occurring hammerheads both in its very slow cleavage rate, and in some aspects of its structure. One striking structural difference is that an additional helix is predicted that may be part of an unusual pseudoknot containing three stacked helices. Nucleotide substitutions that prevent formation of the additional helix and favor the hammerhead increased the self-cleavage rate up to 400-fold. Compensatory substitutions, predicted to restore the additional helix, reduced the self-cleavage rate by an extent proportional to the calculated stability of the helix. Partial digestion of the RNA with structure-sensitive nucleases supported the existence of the proposed alternative structure in the wildtype sequence, and formation of the hammerhead in the rapidly-cleaving mutants. This tertiary interaction may serve as a molecular switch that controls the rate of self-cleavage and possibly other functions of the satellite RNA.     

Efficient ligation of the Schistosoma hammerhead ribozyme

The hammerhead ribozyme from Schistosoma mansoni is the best characterized of the natural hammerhead ribozymes. Biophysical, biochemical, and structural studies have shown that the formation of the loop-loop tertiary interaction between stems I and II alters the global folding, cleavage kinetics, and conformation of the catalytic core of this hammerhead, leading to a ribozyme that is readily cleaved under physiological conditions. This study investigates the ligation kinetics and the internal equilibrium between cleavage and ligation for the Schistosoma hammerhead. Single turnover kinetic studies on a construct where the ribozyme cleaves and ligates substrate(s) in trans showed up to 23% ligation when starting from fully cleaved products. This was achieved by an approximately 2000-fold increase in the rate of ligation compared to a minimal hammerhead without the loop-loop tertiary interaction, yielding an internal equilibrium that ranges from 2 to 3 at physiological Mg2+ ion concentrations (0.1-1 mM). Thus, the natural Schistosoma hammerhead ribozyme is almost as efficient at ligation as it is at cleavage. The results here are consistent with a model where formation of the loop-loop tertiary interaction leads to a higher population of catalytically active molecules and where formation of this tertiary interaction has a much larger effect on the ligation than the cleavage activity of the Schistosoma hammerhead ribozyme.     

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.     

Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay

Hammerhead ribozymes are self-cleaving RNA molecules capable of regulating gene expression in living cells. Their cleavage performance is strongly influenced by intra-molecular loop–loop interactions, a feature not readily accessible through modern prediction algorithms. Ribozyme engineering and efficient implementation of ribozyme-based genetic switches requires detailed knowledge of individual self-cleavage performances. By rational design, we devised fluorescent aptamer-ribozyme RNA architectures that allow for the real-time measurement of ribozyme self-cleavage activity in vitro. The engineered nucleic acid molecules implement a split Spinach aptamer sequence that is made accessible for strand displacement upon ribozyme self-cleavage, thereby complementing the fluorescent Spinach aptamer. This fully RNA-based ribozyme performance assay correlates ribozyme cleavage activity with Spinach fluorescence to provide a rapid and straightforward technology for the validation of loop–loop interactions in hammerhead ribozymes.     

An oligodeoxyribonucleotide that supports catalytic activity in the hammerhead ribozyme domain.

A study of the activity of deoxyribonucleotide-substituted analogs of the hammerhead domain of RNA catalysis has led to the design of a 14mer oligomer composed entirely of deoxyribonucleotides that promotes the cleavage of an RNA substrate. Characterization of this reaction with sequence variants and mixed DNA/RNA oligomers shows that, although the all-deoxyribonucleotide oligomer is less efficient in catalysis, the DNA/substrate complex shares many of the properties of the all-RNA hammerhead domain such as multiple turnover kinetics and dependence on Mg2+ concentration. On the other hand, the values of kinetic parameters distinguish the DNA oligomer from the all-RNA oligomer. In addition, an analog of the oligomer having a single ribonucleotide in a strongly conserved position of the hammerhead domain is associated with more efficient catalysis than the all-RNA oligomer.     

Mutational analyses of the p35-caspase interaction. A bowstring kinetic model of caspase inhibition by p35

Apoptosis is a highly regulated multistep process for programmed cellular destruction. It is centered on the activation of a group of intracellular cysteine proteases known as caspases. The baculoviral p35 protein effectively blocks apoptosis through its broad spectrum caspase inhibition. It harbors a caspase recognition sequence within a highly protruding reactive site loop (RSL), which gets cleaved by a target caspase before the formation of a tight complex. The crystal structure of the post-cleavage complex between p35 and caspase-8 shows that p35 forms a thioester bond with the active site cysteine of the caspase. The covalent bond is prevented from hydrolysis by the N terminus of p35, which repositions into the active site of the caspase to eliminate solvent accessibility of the catalytic residues. Here, we report mutational analyses of the pre-cleavage and post-cleavage p35/caspase interactions using surface plasmon resonance biosensor measurements, pull-down assays and kinetic inhibition experiments. The experiments identify important structural elements for caspase inhibition by p35, including the strict requirement for a Cys at the N terminus of p35 and the rigidity of the RSL. A bowstring kinetic model for p35 function is derived in which the tension generated in the bowstring system during the pre-cleavage interaction is crucial for the fast post-cleavage conformational changes required for inhibition.