Chapter category: RNA
Aminoglycosides and Cleavage of the Hairpin Ribozyme
RNA-Binding Antibiotics
Edited by: Renee SchroederISBN: 1-58706-012-4
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Chapter authors:
David J. Earnshaw and Michael J. Gait
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The hairpin ribozyme belongs to a class of small naturally occurring ribozymes which includes the hammerhead, the hepatitis delta virus and the Neurospora VS ribozyme that in each case generates identical cleavage products that contain 5'hydroxyl and 2',3'cyclic phosphate termini. However, the structures of these small ribozymes and the mechanistic pathways to achieve such cleavages are quite distinct from one another. For recent reviews see refs. 14.
The hairpin ribozyme (reviewed in refs. 58) occurs in the negative strand of the satellite RNA associated with the tobacco ringspot virus (sTRSV) and forms part of a 4way helical junction (Fig. 1a).911 The minimum catalytic motif consists of just two of these helical domains and comprises in total approximately 64 nucleotides which can be configured for cleavage in trans, such that a substrate strand is cleaved by a ribozyme strand. The hairpin that is used within our laboratory is based on this minimal ribozyme but where the ribozyme part is constructed from two separate strands in order to facilitate chemical synthesis (Fig. 1b).1215 Sitespecific cleavage occurs in domain A and, as for most other wellknown small ribozymes, is enhanced dramatically by the presence of magnesium ions.
Both the 4way junction and hinged forms of the hairpin ribozyme achieve their active configuration through an initial docking of domains A and B in an antiparallel alignment. An alternative inactive conformation is also possible where the two domains are stacked in an extended structure. The coexistence of these two forms is responsible for the observed biphasic kinetics, consisting of a fast phase (kcat ~ 0.2 min1) and a subsequent slower phase.16,17 The docking of domains A and B has been monitored by fluorescence resonance energy transfer (FRET) in both the 4way junction and the hinged forms of the hairpin ribozyme.10, 18 This involved the attachment of fluorescent dyes to each domain and subsequent measurement of FRET induced by certain metal ions upon folding. The docking process was found to be faster (~ 0.5 min1) in the presence of metal ions such as magnesium, calcium and strontium than cleavage and is thus not ratelimiting.18 In the case of the 4way junction, efficient docking requires only micromolar concentration of magnesium ions, but millimolar concentrations of the metal ions are necessary for cleavage.10 Additional interactions between the loops facilitate interdomain folding, for example the recently proposed "ribose zipper" between pairs of neighboring hydroxyl groups on the two domains.14 Since docking is fast, the ratelimiting step in the cleavage pathway must occur subsequently.
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