Hairpin ribozyme

Both cleavage and end joining reactions are mediated by the ribozyme motif, leading to a mixture of interconvertible linear and circular satellite RNA molecules.

[7] These newer sequences were hypothesized[7] to occur in organisms that, like those containing the three previously found hairpin ribozymes, use single-stranded, circular RNA genomes.

[9] Using these artificially derived sequences, a trans-acting ribozyme was developed that can catalyze the cleavage of multiple substrate molecules.

This strategy was important in that it allowed investigators to (i) apply biochemical methods for enzymatic analysis, (ii) conduct experiments to identify essential structural elements of the ribozyme-substrate complex, and (iii) develop engineered ribozymes that have been used for biomedical applications, including preventing the replication of pathogenic viruses, and the study of the function of individual genes.

Furthermore, in crystal structures of a ribozyme-inhibitor complex and a transition state mimic, it was shown that the three-dimensional architecture splays apart A-1 and G+1, positioning the 2'-OH of A-1 for an in-line nucleophilic attack on the scissile phosphate linkage.

Additionally, G8, A38, and A9 have been suggested to play roles in the catalysis by deprotonating the 2'-OH of A-1, stabilizing the developing negative charge of the pentacoordinate phosphate oxygens, and protonating the 5'-O leaving group of G+1.

[12][13] The minimal hairpin ribozyme-substrate complex folds into a secondary structure that includes two domains, each consisting of two short base paired helices separated by an internal loop.

[15] When the minimal ribozyme-substrate complex is allowed to fold under conditions of low ionic strength, the two domains stack one atop the other, forming an inactive, extended structure that resembles a hairpin.

[17] The structure and activity of the hairpin ribozyme has been explored using a wide range of complementary experimental methods, including nucleotide replacement, functional group substitution, combinatorial selection, fluorescence spectroscopy, covalent crosslinking, NMR analysis and x-ray crystallography.

Secondary structure of a minimal hairpin ribozyme with substrate RNA bound. Circles represent individual nucleotides and lines indicate canonical (Watson-Crick) base pairs
Folding of the hairpin ribozyme in its native tertiary structure. The ribozyme sequence is shown in grey, whilst the substrate sequence is light red. The cleavage and ligation site (dark red) is between nucleotides A-1 and G+1. Important sequences within loops A and B are shown, with black dots indicating non-Watson-Crick interactions between nucleotides. The two catalytic nucleotides are shown in green, and the critical nucleotide C25, which forms a Watson-Crick base pair with G+1 at the reaction site, is shown in blue. [ 2 ]
A representation of the 3D structure of the hairpin ribozyme. [ 14 ]