Cross-linking experiments reveal the presence of novel structural features between a hepatitis delta virus ribozyme and its substrate

RNA group/groupe ARN, Département de biochimie, Faculté de médecine, Université de Sherbrooke, Québec J1H 5N4, Canada.
RNA (Impact Factor: 4.94). 08/2004; 10(7):1059-72. DOI: 10.1261/rna.7230604
Source: PubMed


The kinetic pathway of a trans-acting delta ribozyme includes an essential structural rearrangement involving the P1 stem, a stem that is formed between the substrate and the ribozyme. We performed cross-linking experiments to determine the substrate position within the catalytic center of an antigenomic, trans-acting, delta ribozyme. Substrates that included a 4-thiouridine either in position -1, +4, or +8 (i.e., adjacent to the cleavage site, or located either in the middle of or at the 3'-end of the P1 stem, respectively) were synthesized and shown to be efficiently cleaved. Examination of the cross-linking conditions, the use of various mutated ribozymes, as well as the probing and characterization of the resulting ribozyme-substrate complexes, revealed several new features of the molecular mechanism: (1) the close proximity of several bases between nucleotides of the substrate and ribozyme; (2) the active ribozyme-substrate complex folds in a manner that docks the middle of the P1 stem on the P3 stem, while concomitantly the scissile phosphate is in close proximity to the catalytic cytosine; and, (3) some complexes appear to be compatible with being active intermediates along the folding pathway, while others seem to correspond to misfolded structures. To provide a model representation of these data, a three-dimensional structure of the delta ribozyme was developed using several RNA bioinformatic software packages.

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Available from: Jonathan Ouellet, Jul 18, 2014
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    • "C9-G35 and U8-A36) tend to become more accessible along the folding pathway. This observation is in agreement with enzymatic probing showing the same behavior of the stem I (Ouellet and Perreault, 2004). In all of the SHAPE experiments, the bands corresponding to both the pseudoknot I.I and the homopurine located at the top of stem IV have strong intensities, suggesting that these base pairs are not always formed and that this region is probably highly dynamic. "
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    ABSTRACT: Both the role and the interacting partners of an RNA molecule can change depending on its tertiary structure. Consequently, it is important to be able to accurately predict the complete folding pathway of an RNA molecule. The hepatitis delta virus (HDV) ribozyme is a small catalytic RNA with the greatest number of folding intermediates making it the model of choice with which to address this problem. The tertiary structures of the known putative intermediates along the folding pathway of the HDV ribozyme were predicted using the Macromolecular Conformations Symbolic programming (MC-Sym) software. The structures obtained by this method received physical support from Selective 2'-Hydroxyl Acylation analyzed by Primer Extension (SHAPE). The analysis of these structures elucidated several features of the HDV ribozyme. In addition, this report represents an application for MC-Sym that permits progression one step further toward the computer prediction of an RNA molecule-folding pathway.
    Structure 12/2010; 18(12):1608-16. DOI:10.1016/j.str.2010.09.024 · 5.62 Impact Factor
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    • "that is single-stranded in the absence of substrate, a fact that is well supported by published data (e.g., see Ouellet and Perreault 2004), are not hydrolyzed in the off state, suggesting that they were either in a helical region or not accessible (Fig. 4A). This might result from an interaction between nucleotides 1 to 7 of the aptamer domain and nucleotides 33 to 39 of the ribozyme domain as, with the exception of one residue, they are complementary. "
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    ABSTRACT: Hepatitis delta virus ribozyme folds into a tightly packed tertiary structure. However, unlike other ribozymes, it does not appear to be able to follow alternative folding pathways. Molecular engineering of the hepatitis delta virus ribozyme led to the development of a ribozyme possessing an endoribonuclease activity that is under the control of a G-quadruplex structure (i.e., a G-quartzyme). This latter species represents an entirely new class of ribozyme. Mutants of this ribozyme were then generated in order to shed light on the modulation of the cleavage activity caused by the presence of the G-quadruplex structure. Kinetic characterization of the G-quartzyme was performed under various single turnover conditions. It was found to be active only in the presence of potassium cations that act as counter ions in the positioning of the four coplanar guanines that form the building block of the G-quadruplex structure. The G-quartzyme behaves as an allosteric ribozyme, with the potassium cations acting as positive effectors with a Hill coefficient of 2.9 +/- 0.2. The conformation transition caused by the presence of the potassium ions is supported by enzymatic and chemical probing of both the inactive (off) and active (on) structures. This study shows that it is possible to interfere with the tight structure of the hepatitis delta virus ribozyme by adding an unusual, stable structure. To our knowledge, the G-quartzyme is the sole ribozyme that exhibits a monovalent cation-dependent activity.
    RNA 07/2008; 14(6):1018-25. DOI:10.1261/rna.963908 · 4.94 Impact Factor
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    • "jo/Delta_Movie.html. These intermediates, although not at an atomic resolution , can be seen as being more than just a plausible set of conformations along the folding pathway as they represent key interactions and structural features reported by various laboratories (e.g., see Nishikawa and Nishikawa 2000; Pereira et al. 2002; Tanaka et al. 2002; Ke et al. 2004; Ouellet and Perreault 2004). Moreover, this animation provides the opportunity to locate the most probable places of these structural features along the folding pathway. "
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    ABSTRACT: With the goal of gaining insight into the tertiary structure of the hepatitis delta virus ribozyme, cross-linking experiments using 4-thiouridine residues introduced in either the 5'-end portion of the substrate, or at seven strategic positions within the ribozyme, were performed. Mapping of the newly formed covalent bonds in cross-linked species obtained under various conditions, as well as using several mutated ribozymes, permitted monitoring of the formation of the ribozyme-substrate complex as the ribozyme proceeded along the folding pathway. In order to aid visualization of the tertiary structure transformation, an in silico animation of the "on" folding pathway was developed. In combination with those of the cleavage assays of structured substrates, these data shed light on the key contribution of the L3 loop in the formation of an active tertiary complex.
    RNA 02/2007; 13(1):44-54. DOI:10.1261/rna.263407 · 4.94 Impact Factor
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