HDV ribozymes

The hepatitis delta virus (HDV) ribozyme is part of the circular single stranded RNA genome of the hepatitis delta virus which consists of a total of 1700 nucleotides. The HDV ribozyme is required for the processing of multimers of the genomic linear RNA transcripts to unit length by catalyzing a transesterification reaction that results in self cleavage [23]. 

Another naturally occurring ribozyme which catalyzes phosphodiester transfer reactions is the hairpin ribozyme. The hairpin ribozyme has been the subject of a number of excellent review articles [24,25]. Several independent studies performed recently have indicated that the hairpin ribozyme has an interesting feature which distinguishes it from the aforementioned ribozymes mechanistically While the HHR, the group I intron, the HDV ribozyme and many other ribozymes that we are going to meet in this review are metalloenzymes and require divalent metal ions in their active sites for functional group activation, divalent metals ions only play a passive role (they are mainly required for cor-... 

Fig. 1A-F The two-dimensional structures of various ribozymes. The ribozyme or intron portion is printed in black. The substrate or exon portion is printed in gray. Arrows indicate sites of cleavage by ribozymes A (left) the two-dimensional structure of a hammerhead ribozyme and its substrate. Outlined letters are conserved bases that are involved in catalysis right) The y-shaped structure of the hammerhead ribozyme-sub-strate complex B-F the two-dimensional structures of a hairpin ribozyme, the genomic HDV ribozyme, a group I ribozyme from Tetrahymena, a group II ribozyme from S. cer-evisiae (aiy5), and the ribozyme of RNase P from E. coli...

HDV ribozymes are derived from the genomic and the antigenomic RNAs of hepatitis delta virus [99-102]. Studies by three groups have revolutionized our understanding of the mechanism of HDV ribozyme-catalyzed reactions [28, 103, 104]. They demonstrated recently that an intramolecular functional group, namely N3 at Gye in the antigenomic HDV ribozyme and N3 at C75 in the genomic HDV ribozyme, can, in fact, act as a true catalyst. However,... 

A novel finding related to the mechanism of catalysis by the genomic HDV ribozyme is that the pKa of C75 is perturbed to neutrality in the ri-bozyme-substrate complex and, more importantly, that C75 acts as a general acid catalyst in combination with a metal hydroxide which acts as a general base catalyst (Fig. 9A) [105]. The discovery of this phenomenon provided the first direct proof that a nucleobase can act as an acid/base catalyst in RNA. As a result, as shown by the solid curve in Fig. 9B, the curve that represents the dependence on the pH of the self-cleavage of the precursor genomic HDV ribozyme has a slope of unity at pH values that are below 7 (the activity increases linearly as the pH increases, with a slope of +1). Then, at higher pH values, the observed rate constant is not affected by the pH. 

All the available data supports the HDV ribozyme-catalyzed reaction mechanism shown in Fig. 9C. In this model, the donation of a proton is favored and the model involves an acid with an optimal pKa of 7 under physi-... 

Nishikawa, S., Suh, Y.A., Kumar, P.K., Kawakami, J., Nishikawa, F. and Taira, K. (1992) Identification of important bases for the self-cleavage activity at two single-stranded regions of genomic HDV ribozyme. Nucleic Acids Symp. Sen. 27,41—42. 

In July 2000 Bartel and coworkers published the impressive example of a single RNA sequence that can adopt either of two ribozyme (Box 22) folds and catalyze two different reactions (Figure 1.1.1) [8]. One reaction is cleavage of RNA catalyzed by the hepatitis delta vims (HDV) ribozyme, which assists the replication of HDV viral RNA. The other is RNA ligation catalyzed by the class III ligase ribozyme, an activity obtained in the laboratory in in-vitro selection experiments. The two ribozyme folds are completely different and do not have a single base pair in common. Importantly, minor variants of this sequence are highly active for one or other catalytic activity and can be accessed from the prototype by a few nucleotide mutations only. 

The hepatitis delta virus (HDV) ribozyme is a member of the class of small ribozymes and functions as a self-cleaving RNA sequence critical to the replication of the virus RNA genome (1, 8, 40). HDV ribozymes are proposed to employ several catalytic strategies that include an important example of general acid/base catalysis that involves a specific cytosine residue in the active site. Indeed, a milestone in our understanding of RNA catalysis was the observation that HDV and other small ribozymes could function in the absence of divalent metal ion cofactors, provided that high (molar) concentrations of monovalent ions are present (41, 42). These high monovalent ion concentrations are believed to stabilize the active RNA conformation, which implies that the primary role of divalent metal ions is in structural stabilization (42). 

Figure 5 Proposed acid/base catalytic interactions in the HDV ribozyme active site. The cleavage site for the HDV ribozyme is shown with the nucleotide 5 to the site of bond cleavage shown in red and the nucleotide 3 to that site shown in blue. Two proposed mechanisms for the function of the C76 cytisine nucleobase and a hydrated active site metal ion are shown in which C76 acts as either an acid (left) or base (right) as described in the text.

The HDV ribozyme, however, is only one example of the class of small ribozymes that catalyze self-cleavage, which includes the hammerhead, hairpin, VS, and glmS riboswitch ribozymes (1, 2). Like the mechanistic detail described above, crystal structures have been solved for several modified versions of these ribozymes, and the structures thus derived provide candidate... 

Wadkins TS, Shih I, Perrotta AT, Been MD. A pH-sensitive RNA tertiary interaction affects seh-cleavage activity of the HDV ribozymes in the absence of added divalent metal ion. J. Mol. Biol. 2001 305 1045-1055. 

The mechanism whereby neomycin B inhibits ribozyme fimction has yet to be elucidated. However, existing results [156] suggest a competitive inhibition of this catalytic RNA by neomycin B, implying that this antibiotic inhibits HDV ribozyme cleavage by disruption of the divalent cationic binding near the catalytic core of the ribozyme. 

In hairpin ribozyme, the nucleobase of G8 is immediately adjacent to the attacking 2 -OH of Al and could play a direct role in catalysis. The HDV ribozyme provides a well-characterized example of nucleobase catalysis. The structure of the ribozyme-product complex has been solved by crystallography (Ferre-d Amare et al., 1998), revealing a deep cleft containing the site of cleavage. It also contains an essential cytosine base (C75 in the... 

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