Hairpin Ribozyme: Difference between revisions

The <scene name='56/560862/Ribozyme_substrate_and_protein/1'>hairpin ribozyme</scene> is a member of a small family of RNA endonucleases that includes hammerhead, hepatitis delta, and Neurospora VS.<ref name="Shippy">PMID: 10554775</ref>  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 name="Shippy"/><ref>PMID: 10715200</ref>

The hairpin ribozyme was discovered in the negative strand of the tobacco ringspot virus (TRSV) satellite RNA.<ref>PMID: 22131309</ref>  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.<ref name="Walter">PMID: 9667918</ref>

<scene name='56/560862/Domain_a_and_domain_b/1'>A (green) and B (yellow)</scene>.  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 name="Walter"/>

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.<ref name="Rupert">PMID: 12376595</ref>

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 <scene name='56/560862/Scissile_phosphate/1'>scissile phosphate</scene> 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.<ref name="Rupert"/>

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 <scene name='56/560862/G8_and_a9/1'>G8, A9</scene>, 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.<ref name="Rupert"/>