Hairpin Ribozyme

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The hairpin ribozyme (red) with substrate (blue) and ribonucleoprotein (green) 1m5k

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The is a member of a small family of RNA endonucleases that includes hammerhead, hepatitis delta, and Neurospora VS.[1] Endonucleases are enzymes that cleave phosphodiester bonds within polynucleotide chains. This group of endonucleases cleave RNA substrates in a reversible reaction that generates a 2', 3'-cyclic phosphate and a 5'-hydroxyl termini.[1][2]

The hairpin ribozyme was discovered in the negative strand of the tobacco ringspot virus (TRSV) satellite RNA.[3] Study of hairpin ribozyme reaction mechanisms provided early evidence that ribozymes are able to exploit a variety of strategies, just like protein enzymes. But, the hairpin ribozyme has a unique characteristic. Unlike other ribozymes, the hairpin ribozyme does not require metal ions for cleavage or ligation of substrate RNA, though it does use metal ions to facilitate domain interactions.[4]


The secondary structure of the hairpin ribozyme contains two independently folding domains, called . In each domain there is an internal loop flanked by two helices (H1 and H2 in domain A and H3 and H4 in domain B). The RNA substrate is bound in domain A through Watson-Crick base pairs in H1 and H2. Once bound to domain A, the substrate is reversibly cleaved. Linkers of varying lengths were inserted between the 5' end of the substrate and the 3' end of the ribozyme in order to test what proximity is preferred by the two domains. The results of the test showed that the two domains prefer to be relatively close to one another and use H2 and H3 as a sort of hinge. In the naturally occurring hairpin ribozyme, this hinge is occupied by a four-way junction, which is believed to regulate inter-domain interactions by alternative stacking of helices.[4]


In the transition state, the hairpin ribozyme demonstrates coordinate bonding. The substrate binds to the 2' and 3' oxygens of nucleotide -1 and the 5' oxygen of nucleotide +1. This bonding creates a large amount of electron density, which is very important to the mechanism. If even one of the coordinate bonds is absent, the electron density feature is absent as well. When the hairpin ribozyme is bound to a substrate that is entirely RNA, the electron density is made up of a mixture of the cleaved and ligated substrate. This is because the ribozyme catalyzes both the cleavage reaction and the reverse ligation.[5]

Crystal structures of the transition state, precursor, and product indicate that the active site remains in a fairly fixed position. The lone motion occurs between the and the ribose of nucleotide -1. The ribose undergoes a change in puckering when its 2' oxygen attacks the phosphate and a five membered 2', 3' phosphate is formed.[5]

Hydrogen bonding is a major contributor to the catalytic process. In the precursor, the ribozyme forms two hydrogen bonds. One is between the nucleobase of G8 and the 2'-OH nucleophile and the other is between the nucleobase of G8 and one of the phosphate oxygens. In the transition state, five hydrogen bonds are formed. Those H-bonds occur between the nucleobases of , and A38 and the oxygens of the substrate. In the product, the nucleobases of G8 and A38 make three hydrogen bonds. Two are formed to the cyclic phosphate and the other is to 5'-OH leaving group.[5]


There are at least four steps in the reaction pathway of the hairpin ribozyme. They are: (1) substrate binding to ribozyme, (2) cleavage in the ribozyme-substrate complex, (3) release of 5' products, and (4) release of 3' products. The rates and equilibrium constants of these individual steps have been studied. Substrate binding by the naturally occurring hairpin ribozyme can reach a minimum of 6x106 M-1 min-1 and modified versions can reach maximums of 5x108 M-1 min-1. The cleavage rate is significantly higher than the dissociation rate, meaning cleavage of bound substrate is highly favored over dissociation. Tests have also shown that the hairpin ribozyme has a biphasic cleavage. The fast phase is due to the ribozyme-substrate complex being folded correctly and the slow phase is due to an inactive conformer with H2 and H3 coaxially stacked.[6]

3D structures of ribozyme



  1. 1.0 1.1 Shippy R, Lockner R, Farnsworth M, Hampel A. The hairpin ribozyme. Discovery, mechanism, and development for gene therapy. Mol Biotechnol. 1999 Aug;12(1):117-29. PMID:10554775 doi:
  2. Fedor MJ. Structure and function of the hairpin ribozyme. J Mol Biol. 2000 Mar 24;297(2):269-91. PMID:10715200 doi:
  3. Muller S, Appel B, Krellenberg T, Petkovic S. The many faces of the hairpin ribozyme: structural and functional variants of a small catalytic RNA. IUBMB Life. 2012 Jan;64(1):36-47. doi: 10.1002/iub.575. Epub 2011 Nov 30. PMID:22131309 doi:
  4. 4.0 4.1 Walter NG, Burke JM. The hairpin ribozyme: structure, assembly and catalysis. Curr Opin Chem Biol. 1998 Feb;2(1):24-30. PMID:9667918
  5. 5.0 5.1 5.2 Rupert PB, Massey AP, Sigurdsson ST, Ferre-D'Amare AR. Transition state stabilization by a catalytic RNA. Science. 2002 Nov 15;298(5597):1421-4. Epub 2002 Oct 10. PMID:12376595 doi:
  6. Esteban JA, Banerjee AR, Burke JM. Kinetic mechanism of the hairpin ribozyme. Identification and characterization of two nonexchangeable conformations. J Biol Chem. 1997 May 23;272(21):13629-39. PMID:9153212

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