Hairpin Ribozyme
From Proteopedia
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<StructureSection load='1m5k' size='350' side='right' caption='Crystal Structure of the Hairpin Ribozyme in the catalytically-active conformation (PDB entry [[1m5k]])' scene=''> | <StructureSection load='1m5k' size='350' side='right' caption='Crystal Structure of the Hairpin Ribozyme in the catalytically-active conformation (PDB entry [[1m5k]])' scene=''> | ||
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The hairpin ribozyme is a member of a small family of RNA endonucleases that includes hammerhead, hepatitis delta, and Neurospora VS. 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.<ref>PMID: 10715200</ref><ref>PMID: 10554775</ref> | The hairpin ribozyme is a member of a small family of RNA endonucleases that includes hammerhead, hepatitis delta, and Neurospora VS. 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.<ref>PMID: 10715200</ref><ref>PMID: 10554775</ref> | ||
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==Structure== | ==Structure== | ||
In the transition state, the <scene name='56/560862/Crystal_structure/1'>hairpin ribozyme</scene> | In the transition state, the <scene name='56/560862/Crystal_structure/1'>hairpin ribozyme</scene> | ||
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The secondary structure of the hairpin ribozyme contains two independently folding domains, called A and B. 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.<ref>PMID: 9667918</ref> | The secondary structure of the hairpin ribozyme contains two independently folding domains, called A and B. 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.<ref>PMID: 9667918</ref> | ||
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Contents |
Hairpin Ribozyme Overview
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Structure
In the transition state, the
The secondary structure of the hairpin ribozyme contains two independently folding domains, called A and B. 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.[5]
Kinetics
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]
References
- ↑ Fedor MJ. Structure and function of the hairpin ribozyme. J Mol Biol. 2000 Mar 24;297(2):269-91. PMID:10715200 doi:http://dx.doi.org/10.1006/jmbi.2000.3560
- ↑ 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:http://dx.doi.org/10.1385/MB:12:1:117
- ↑ 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:http://dx.doi.org/10.1002/iub.575
- ↑ Walter NG, Burke JM. The hairpin ribozyme: structure, assembly and catalysis. Curr Opin Chem Biol. 1998 Feb;2(1):24-30. PMID:9667918
- ↑ Walter NG, Burke JM. The hairpin ribozyme: structure, assembly and catalysis. Curr Opin Chem Biol. 1998 Feb;2(1):24-30. PMID:9667918
- ↑ 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
