Self‐Cleaving Ribozymes of Hepatitis Delta Virus RNA

Abstract

Hepatitis delta virus (HDV) is a small single-stranded RNA satellite of hepatitis B virus. Although it is a human pathogen, it shares a number of features with a subset of the small plant satellite RNA viruses, including self-cleaving sequences in the genomic and antigenomic sequences of the viral RNA. The selfcleaving sequence is critical to viral replication and is thought to function as a ribozyme in vivo to process the products of rolling-circle replication to unit-length molecules. A divalent cation is required for cleavage and while a structural role is implicated for metal ions, a more direct role for a metal ion in catalysishas not yet been proven. A minimal natural ribozyme sequence with proficient in viiro self-cleavage activity is about 85 nucleotides long and adopts a secondary structure with four paired regions (Pl-P4).The two pairings that define the 5' and 3' boundaries of the ribozyme, PI and P2, form an atypical pseudoknot arrangement. This secondary structure places a number of constraints on the possible tertiary folding of the sequence, which together with chemical probing, photo-cross-linking, mutagenesis and computer-assisted modeling provides clues to the three-dimensional structure. The data are consistent with a model in which the cleavage site, located at the 5' end of PI, is in close proximity to three singlestranded regions, consisting of a hairpin loop at the end of P3 and two sequences joining P1 to P4 and P4 to P2. While the natural forms of the HDV ribozymes appear to be prone to misfolding, biochemical and mutagenesis studies from a number of laboratories has allowed the production of trans-acting ribozymes and smaller more active cis-acting ribozymes, both of which will aid in further mechanistic and structural studies of this RNA.

tRNA-dependent cleavage of the ColE1 plasmid-encoded RNA I

“…hammerhead motif ribozyme (Birikh et al, 1997); hairpin motif ribozyme (Fedor, 2000); hepatitis delta virus ribozyme (Been & Wickham, 1997); varkud satellite (VS) ribozyme (Collins, 2002)], and self-splicing RNAs [for example: group I introns (Schmidt et al, 1992) and group II introns (Jacquier, 1996)]. (c) Fig.…”

HDV Family of Self-Cleaving Ribozymes

“…[41][42][43][44] When this motif is properly positioned in the genomic ribozyme, the N4 of the conserved C75 residue forms two hydrogen bonds, one with a nonbridging oxygen atom of the phosphate backbone linking the C21 and C22 residues that form the 5 0 side of the P1.1 helix, and a second to the 2 0 OH of U20. [34][35][36] These interactions lock the J4/2 region into the active site of the ribozyme.…”

“…44 Formation of a properly base-paired P3 is essential for catalysis; therefore, the observed conservation may instead reflect the rarity of two compensatory mutations occurring in this region in a single replicative event. 41 Unlike the P3 helix, the P2 helix varies greatly in both nucleotide composition and length among the HDV-like motifs. In the R2 ribozymes, this region is potentially longer than the six to eight base-pair norm, suggesting, in conjunction with its high level of conservation, that it has an important role in catalysis or retrotransposition.…”

Kinetic Analysis of δ Ribozyme Cleavage

“…Trans-acting δ ribozymes of either polarity have been designed and used almost exclusively to study the impact of mutations on cleavage activity (2). In general, trans-acting ribozymes were shown to cleave at a slower rate than the corresponding self-cleaving RNA strand.…”