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.     

Bis-aptazyme sensors for hepatitis C virus replicase and helicase without blank signal

The fusion molecule (i.e. aptazyme) of aptamer and hammerhead ribozyme was developed as in situ sensor. Previously, the hammerhead ribozyme conjugated with aptamer through its stem II module showed a significant blank signal by self-cleavage. To reduce or remove its self-cleavage activity in the absence of target molecule, rational designs were attempted by reducing the binding affinity of the aptazyme to its RNA substrate, while maintaining the ribonuclease activity of the aptazyme. Interestingly, the bis-aptazymes which comprise the two aptamer-binding sites at both stem I and stem III of the hammerhead ribozyme showed very low blank signals, and their ratios of reaction rate constants, i.e. signal to noise ratios, were several tens to hundred times higher than those of the stem II-conjugated bis-aptazymes. The reduction in the blank signals seems to be caused by a higher dissociation constant between the main strand of the bis-aptazyme and its substrate arising from multi-point base-pairing of the bis-aptazymes. The bis-aptazymes for HCV replicase and helicase showed high selectivity against other proteins, and a linear relationship existed between their ribozyme activities and the target concentrations. In addition, a bis-aptazyme of dual functions was designed by inserting both aptamers for HCV replicase and helicase into the stem I and stem III of hammerhead ribozyme, respectively, and it also showed greater sensitivity and specificity for both proteins without blank signal.     

A catalytic 13-mer ribozyme.

A 13-mer oligoribonucleotide can act as a ribozyme for the specific self-cleavage of a 41-mer oligoribonucleotide substrate in the presence of Mg2+. The two sequences involved correspond to the self-cleavage hammerhead structure of the virusoid of lucerne transient streak virus. The Michaelis-menten kinetic parameters for the reaction were; Km 1.3 microM, Vmax 0.012 microM min-1, kcat 0.5 min-1. The 13-mer RNA is the smallest ribozyme so far reported. A DNA analogue of the 13-mer can not substitute for the RNA in the reaction.     

The Global Conformation of an Active Hammerhead RNA during the Process of Self-cleavage

The RNA “hammerhead” domain is a small element of secondary structure found in the genomes of certain plant pathogens. It possesses a core of conserved sequence at the conjunction of three helix stems, and is capable of undergoing self-cleavage in the presence of divalent cations. Both crystallographic and solution studies suggest that the domain is highly structured, with the three stems assuming a Y-shaped global conformation; however, such studies have employed either RNA analogues that were catalytically inactive, or conditions of temperature and pH for which rates of self-cleavage are slow. Thus, it was unknown whether such species represented the principal conformers during the cleavage process itself. In order to address this issue, a series of time- resolved, transient electric birefringence measurements was conducted in an effort to define the global conformation of an RNA hammerhead in real time throughout the process of self-cleavage. The current study demonstrates that the angular relationship between the two helices that flank the cleavage center is essentially unchanged between the pre-cleavage and post-cleavage forms. Moreover, despite the fact that at least one kinetic intermediate is formed during the self-cleavage reaction, there is no evidence for the existence of a significant population of intermediates with altered global conformation during cleavage. Thus, any conformational isomerism that may occur is likely to be relatively localized to the active center. Finally, it was observed that sequence elements lying outside of the conserved region, at the base of stem I, influence interhelix geometry. The current results are consistent with a structural model in which the active center possesses similar conformations pre-cleavage and post-cleavage. Such a model would help to explain the significant rate of reversal of the cleavage reaction (self-ligation).     

Self-cleavage of p2Sp1 RNA with Mg2+ and non-ionic detergent (Brij 58)

The precursor of an RNA molecule from T4-infected E. coli cells (p2Sp1 RNA) has the capacity to cleave itself at specific positions [(UpA (139-140) and CpA (170-171)], within a putative loop and stem structure. This sequence-specific cleavage requires at least a monovalent cation and non-ionic detergents. We studied the self-cleavage reaction of an RNA fragment (GUUUCGUACAAAC) (R1) with the sequence corresponding to the p2Sp1 RNA in the presence of Mg2+ and non-ionic detergents. It requires Mg2+ and is aided by a non-ionic detergent, Brij 58. The cleavage reaction is time, temperature, and pH-dependent. The cleavage occurs at the phosphodiester bond between UpA and CpA on the RNA fragment (GUUUCGUACAAAC) (R1). Furthermore, the maximum of cleavage of R1 occurs at a very low Mg2+ concentration (< or = 5 mM).     

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.     

The stem hairpin loop structure of p2Sp1 RNA is required for RNA-cleaving activity

We studied the hairpin-loop structure of an RNA fragment (GUUUCGUACAAAC) (R13) with the sequence corresponding to the self-cleavage domain in the precursor of an RNA molecule from bacteriophage T4-infected Escherichia coli cells (p2Sp1 RNA). In order to determine the influence of the hairpin-loop structure on these sequence-specific cleavage reactions, we have synthesized oligoribonucleotides containing hairpin-loop, double-helical stem-loop, and single-stranded RNA structures. The cleavage was affected by the hairpin-loop structure. Furthermore, the helix-stem, which retains the thermodynamically extrastable stem hairpin-loop structures, is also important for the cleavage activity. However, the thermodynamically extrastable helix-stem structure reduced the cleavage activity of the adjacent UA and CA sequences at the helix-stem site. For the cleavage reactions of the RNA cleavage products, the R6 (ACAAAC), R7 (GUUUCGU), and R9 (GUUUCGUAC) mers from the parent RNA, R13 (GUUUCGUACAAAC), a very slight amount of cleavage product (2%) from the RNA 9 was observed, but no reaction occurred for the R6 and R7. We also describe the influences of the sequences (UA and CA) on the cleavage activity.     

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.     

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.     

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.