page_title: Hammerhead ribozyme
section_title: Structure and catalysis
hierarchical_section_title: Hammerhead ribozyme / Structure and catalysis
caption_attribution_description: Hammerhead Ribozyme active site
context_page_description: The hammerhead ribozyme is an RNA motif that catalyzes reversible cleavage and ligation reactions at a specific site within an RNA molecule. It is one of several catalytic RNAs known to occur in nature. It serves as a model system for research on the structure and properties of RNA, and is used for targeted RNA cleavage experiments, some with proposed therapeutic applications. Named for the resemblance of early secondary structure diagrams to a hammerhead shark, hammerhead ribozymes were originally discovered in two classes of plant virus-like RNAs: satellite RNAs and viroids. They have subsequently been found to be widely dispersed within many forms of life.
  The self-cleavage reactions, first reported in 1986, are part of a rolling circle replication mechanism. The hammerhead sequence is sufficient for self-cleavage and acts by forming a conserved three-dimensional tertiary structure.
context_section_description: The tertiary interactions in the full-length hammerhead ribozyme stabilize what strongly appears to be the active conformation. The nucleophile, the 2'-oxygen of the cleavage-site nucleotide, C17, is aligned almost perfectly for an in-line attack (the SN2(P) reaction). G12 is positioned within hydrogen bonding distance of this nucleophile, and therefore would be able to abstract a proton from the 2'-oxygen if G12 itself becomes deprotonated. The 2'-OH of G8 forms a hydrogen bond to the 5'-leaving group oxygen, and therefore potentially may supply a proton as negative charge accumulates on the 5'-oxygen of the ribose of A1.1.
  The most likely explanation is then that G12, in the deprotonated form, is the general base, and the ribose of G8 is the general acid. The apparent kinetic pKa of the hammerhead ribozyme is 8.5, whereas the pKa of guanosine is about 9.5. It is possible that the pKa of G12 is perturbed from 9.5 to 8.5 in the hammerhead catalytic core; this hypothesis is currently the subject of intense investigation.
  If the invariant G8 is changed to C8, hammerhead catalysis is abolished. However, a G8C + C3G double-mutant that maintains the G8-C3 base pair found in the full-length hammerhead restores most of the catalytic activity. The 2'-OH of G8 has also been observed to be essential for catalysis; replacement of G8 with deoxyG8 greatly reduces the rate of catalysis, suggesting the 2'-OH is indeed crucial to the catalytic mechanism.
  The close approach of the A9 and scissile phosphates requires the presence of a high concentration of positive charge. This is probably the source of the observation that divalent metal ions are required at low ionic strength, but can be dispensed with at higher concentrations of monovalent cations.
  The reaction thus likely involves abstraction of the 2'-proton from C17, followed by nucleophilic attack upon the adjacent phosphate. As the bond between the scissile phosphorus and the 5'-O leaving group begins to break, a proton is supplied from the ribose of G8, which then likely reprotonates at the expense of a water molecule observed to hydrogen bond to it in the crystal structure.
caption: The active site of the full-length hammerhead ribozyme. G12 is positioned consistent with a role as a general base in the cleavage reaction, and the 2'-OH of G8 is positioned for acid catalysis. Potentially "active" hydrogen bonds are shown as orange dotted lines. The 2'-O of C17 is shown to be aligned for nucleophilic attack along the blue dotted line trajectory.
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