Structure and function of the hairpin ribozyme

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.…”

The effect of cytidine on the structure and function of an RNA ligase ribozyme

“…Time course of the intermolecular ligation reaction catalyzed by the R3C ribozyme+ The 58mer ribozyme was allowed to react with an unlabeled 12mer substrate and labeled 18mer substrate (S), under standard conditions (see Materials and Methods), giving rise to a unique 30mer product (P)+ The ribozyme and 12mer substrate were present at saturating concentrations+ The reaction was sampled at 2, 5, 15, 45, 90, and 120 min+ The products were separated by electrophoresis in a denaturing polyacrylamide gel, an autoradiogram of which is shown+ N1 and/or N2 positions, whereas all of the other nucleotides of the purine-rich bulge are unprotected+ This suggests that the guanosine 59-triphosphate is engaged in a nonstandard pair, perhaps involving hydrogen bonding of its N1 and O6 positions with the N7 and N6 positions of an opposing adenosine+ There are some structural similarities between the R3 ligase and the hairpin ribozyme, both of which catalyze RNA ligation, albeit by a different chemical mechanism+ Both ribozymes contain an A-G sequence flanking the ligation junction and an unpaired 59-GAA-39 sequence on the opposing strand+ Both contain extended double helical domains that meet at a junction and interact through tertiary contacts that help form the active enzyme-substrate complex+ In the case of the hairpin ribozyme, these contacts involve interactions between the bulge loop at the ligation junction and a bulge loop within the other extended helical domain (for recent review, see Fedor, 2000)+ Analogous tertiary interactions may occur among the bulge loops of the R3 ribozyme, helping to stabilize an otherwise poorly constrained structure+ There are two major and conflicting obstacles that must be overcome in the development of a cytidinefree ribozyme+ The first is the difficulty in forming a stable secondary structure in the absence of G•C pairs+ This can be accomplished with longer stem regions based on A•U and G•U pairs+ The second obstacle is the need to avoid alternative, inactive conformations that are likely to occur when every purine (A or G) is complementary to every pyrimidine (U)+ The longer the RNA, as would be required to form a stable secondary structure, the more difficult it will be to avoid alternative conformations+ These two constraints may explain why the R3 ribozyme adopts a simple secondary structure that is formed by a modest number of nucleotides+ Following the introduction of cytidine, the stem regions became more stable and more compact, allowing a more reactive secondary structure to form that was less susceptible to alternative conformations+ There are subtle differences in the secondary structures of the R3 and R3C ribozymes+ Six of the 12 mutations that arose in the R3C ribozyme, including five of the seven added cytidines, stabilize the stem regions by converting A•U or G•U pairs to G•C pairs+ Five other mutations result in remodeling of the bulge loops that lie adjacent to the three-way junction+ The bulge-remodeling mutations alone were highly detrimental to the catalytic activity of the R3 ligase, but together with the stem-stabilizing mutations proved highly beneficial+…”

Dissecting the multistep reaction pathway of an RNA enzyme by single-molecule kinetic “fingerprinting”

“…The hairpin ribozyme catalyzes a reversible site-specific RNA cleavage reaction (4,5). The enzyme consists of two helix-loophelix domains, with the cleavage site located within the substrate strand that makes up half of loop A (Fig.…”