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William G. Scott - One of the best experts on this subject based on the ideXlab platform.
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Structural Simplicity and Mechanistic Complexity in the Hammerhead Ribozyme.
Progress in molecular biology and translational science, 2018Co-Authors: Sara M. O'rourke, William G. ScottAbstract:Natural or full-length Hammerhead Ribozymes are up to 1000-fold more active than their minimal counterparts that lack a complex tertiary interaction that pre-organizes and stabilizes the Ribozyme active site, positioning RNA functional groups to facilitate acid-base catalysis. The recent discovery that a single tertiary contact (an AU Hoogsteen pair) between Stems I and II confers essentially all of the enhanced activity greatly simplifies our understanding of the structural requirements for Hammerhead Ribozyme activity. In contrast, the simplest mechanistic interpretations are challenged with the presentation of more complex alternatives. These alternatives are elucidated and critically analyzed in the context of several of the active Hammerhead Ribozyme structures now available.
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The Hammerhead Ribozyme: Structure, Catalysis, and Gene Regulation
Progress in molecular biology and translational science, 2013Co-Authors: William G. Scott, Horan Lucas, Monika MartickAbstract:The Hammerhead Ribozyme has long been considered a prototype for understanding RNA catalysis, but discrepancies between the earlier crystal structures of a minimal Hammerhead self-cleaving motif and various biochemical investigations frustrated attempt to understand Hammerhead Ribozyme catalysis in terms of structure. With the discovery that a tertiary contact distal from the Ribozyme’s active site greatly enhances its catalytic prowess, and the emergence of new corresponding crystal structures of full-length Hammerhead Ribozymes, a unified understanding of catalysis in terms of the structure is now possible. A mechanism in which the invariant residue G12 functions as a general base, and the 2′-OH moiety of the invariant G8, itself forming a tertiary base pair with the invariant C3, is the general acid, appears consistent with both the crystal structure and biochemical experimental results. Originally discovered in the context of plant satellite RNA viruses, the Hammerhead more recently has been found embedded in the 3′-untranslated region of mature mammalian mRNAs, suggesting additional biological roles in genetic regulation.
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What can the New Hammerhead Ribozyme Structures Teach us About Design
RNA Technologies and Their Applications, 2010Co-Authors: William G. ScottAbstract:The Hammerhead Ribozyme is a small, self-cleaving genomic Ribozyme whose substrate-targeting properties are quite flexible. It catalyzes a phosphodiester backbone cleavage reaction that can be exploited for antisense-type applications in which it is desirable to cleave the target RNA. To better understand the requirements for rational Hammerhead Ribozyme design, the natural history, secondary and tertiary structures, and reaction mechanism are reviewed in detail. Specifically, significant advances in our understanding of how the Hammerhead Ribozyme works have taken place since 2003, rendering previous assumptions about therapeutic Hammerhead Ribozyme design largely obsolete. The requirement for a tertiary contact between Stems I and II to be present in order to achieve a highly active Ribozyme in vivo is described, and design requirements that enable straightforward incorporation of the tertiary contact are explicitly described. This analysis is only possible with crystal structures of two classes of full-length natural Hammerhead Ribozymes that became available in 2006 and 2008.
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role of mg2 in Hammerhead Ribozyme catalysis from molecular simulation
Journal of the American Chemical Society, 2008Co-Authors: Tai-sung Lee, William G. Scott, Monika Martick, Carlos Silva Lopez, George M Giambasu, Darrin M. YorkAbstract:Molecular dynamics simulations have been performed to investigate the role of Mg2+ in the full-length Hammerhead Ribozyme cleavage reaction. In particular, the aim of this work is to characterize the binding mode and conformational events that give rise to catalytically active conformations and stabilization of the transition state. Toward this end, a series of eight 12 ns molecular dynamics simulations have been performed with different divalent metal binding occupations for the reactant, early and late transition state using recently developed force field parameters for metal ions and reactive intermediates in RNA catalysis. In addition, hybrid QM/MM calculations of the early and late transition state were performed to study the proton-transfer step in general acid catalysis that is facilitated by the catalytic Mg2+ ion. The simulations suggest that Mg2+ is profoundly involved in the Hammerhead Ribozyme mechanism both at structural and catalytic levels. Binding of Mg2+ in the active site plays a key str...
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Solvent Structure and Hammerhead Ribozyme Catalysis
Chemistry & biology, 2008Co-Authors: Monika Martick, Tai-sung Lee, Darrin M. York, William G. ScottAbstract:Although the Hammerhead Ribozyme is regarded as a prototype for understanding RNA catalysis, the mechanistic roles of associated metal ions and water molecules in the cleavage reaction remain controversial. We have investigated the catalytic potential of observed divalent metal ions and water molecules bound to a 2 A structure of the full-length Hammerhead Ribozyme by using X-ray crystallography in combination with molecular dynamics simulations. A single Mn(2+) is observed to bind directly to the A9 phosphate in the active site, accompanying a hydrogen-bond network involving a well-ordered water molecule spanning N1 of G12 (the general base) and 2'-O of G8 (previously implicated in general acid catalysis) that we propose, based on molecular dynamics calculations, facilitates proton transfer in the cleavage reaction. Phosphate-bridging metal interactions and other mechanistic hypotheses are also tested with this approach.
Fritz Eckstein - One of the best experts on this subject based on the ideXlab platform.
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selection of Hammerhead Ribozyme variants with low mg2 requirement importance of stem loop ii
ChemBioChem, 2002Co-Authors: Tina Persson, Roland K Hartmann, Fritz EcksteinAbstract:Variants of the Hammerhead Ribozyme with high in trans (intermolecular) cleavage activity at low Mg(2+) concentrations were in vitro selected from a library with 18 nucleotides randomised in the core and in helix II. The most active Hammerhead Ribozyme selected had the same sequence as the consensus Ribozyme in the core but only two base pairs in stem II, G(10.1)-C(11.1) and U(10.2)-A(11.2), and a tetrauridine loop II. This Ribozyme (clone 34) was found to be very active in single-turnover reactions at 1 mM Mg(2+) concentration in the context of several substrates with differences in the lengths of stem I and III, including the well-characterised HH16 substrate and a derivative thereof with a GUA triplet at the cleavage site, as well as a substrate used previously in a related study. For the HH16 substrate, a change of base pair 10.2-11.2 to C-G in stem II further improved activity by about 2.5-fold to 0.8 min(-1) (at 1 mM Mg(2+) concentration, 25 degrees C, pH 7.5). Interestingly, this very active variant was not identified by the selection procedure. Changing loop II from UUUU to GCAA or extension of stem II to three or four base pairs reduced the cleavage rate by 2.0-2.5-fold. Thus, small Hammerhead Ribozymes carrying a tetrauridine loop with two base pairs in stem II represent the most active versions known so far at low Mg(2+) concentrations; single-turnover rates of approximately 1 min(-1) are reached at 25 degrees C and pH 7.5 in monophasic reactions, with endpoints between 75 and 90 %. Such constructs promise to be advantageous for the inhibition of gene expression in vivo.
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The Hammerhead Ribozyme.
Biopolymers, 1999Co-Authors: Fritz Eckstein, Birgit BramlageAbstract:It was 10 years ago that the Hammerhead Ribozyme catalytic RNA motif was first detected in certain small satellite and viroid RNAs (for reviews see Symons 1992 and Bratty et al. 1993). Its two-dimensional representation (Fig. 1) has now been transformed into a three-dimensional model by the application of X-ray crystallography (Pley et al. 1994; Scott et al. 1995) (see also chapter by McKay), fluorescence resonance energy transfer (FRET) measurements (Tuschl et al. 1994), gel electrophoresis (Bassi et al. 1995), and transient electric birefringence measurements (Amiri and Hagerman 1994). In the past decade, the potential of Ribozymes for the inhibition of gene expression has been demonstrated (for a review see Marschall et al. 1994). Particular examples are discussed in the chapters by Arndt and Atkins, Bertrand and Rossi, Sproat, Sun et al., Welch et al., and Usman and Stinchcomb, this Vol. This application has been successfully extended to the study of transgenic animals containing a Hammerhead Ribozyme gene targeted against the gene of interest (Heinrich et al. 1993; Zhao and Pick 1993; Efrat et al. 1994; Larsson et al. 1994; see also L’Huillier, this Vol.).
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recent developments in the Hammerhead Ribozyme field
Nucleic Acids Research, 1998Co-Authors: Narendra K. Vaish, Anilkumar R Kore, Fritz EcksteinAbstract:Developments in the Hammerhead Ribozyme field during the last two years are reviewed here. New results on the specificity of this Ribozyme, the mechanism of its action and on the question of metal ion involvement in the cleavage reaction are discussed. To demonstrate the potential of Ribozyme technology examples of the application of this Ribozyme for the inhibition of gene expression in cell culture, in animals, as well as in plant models are presented. Particular emphasis is given to critical steps in the approach, including RNA site selection, delivery, vector development and cassette construction.
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sequence specificity of the Hammerhead Ribozyme revisited the nhh rule
Nucleic Acids Research, 1998Co-Authors: Anilkumar R Kore, Narendra K. Vaish, Ursula Kutzke, Fritz EcksteinAbstract:The sequence specificity of Hammerhead Ribozyme cleavage has been re-evaluated with respect to the NUH rule. Contrary to previous reports it was found that substrates with GAC triplets were also cleaved. This was established in three different sequence contexts. The rate of cleavage under single turnover conditions was between 3 and 7% that of cleavage 34 of GUC. Specificity of cleavage of substrates containing a central A in the cleavable triplet can be described as NAH, where N can be any nucleotide and H any nucleotide but G. As cleavage 34 of NCH triplets has recently been described, the NUH rule can be reformulated to NHH.
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the structure function and application of the Hammerhead Ribozyme
FEBS Journal, 1997Co-Authors: Klara R Birikh, Paul A Heaton, Fritz EcksteinAbstract:The Hammerhead Ribozyme is one of the smallest Ribozymes known and catalyses the site-specific hydrolysis of a phosphodiester bond. This small Ribozyme is of interest for two reasons. It offers a convenient system to study the structure/function relationship of a nucleotide sequence, and is a potential vehicle for the inhibition of gene expression. The first part of the review summarizes the sequence requirements of the Hammerhead, its three-dimensional structure and the proposed mechanism, in addition to Ribozyme specificity and turnover. The second part of the review focuses on the in vivo application of the Ribozyme. The processes involved in designing Ribozymes for efficient cleavage in vivo are described, together with possible delivery strategies.
David M.j. Lilley - One of the best experts on this subject based on the ideXlab platform.
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RNA folding and misfolding of the Hammerhead Ribozyme.
Biochemistry, 1999Co-Authors: Gurminder S. Bassi, Niels Erik Møllegaard, A. I. H. Murchie, David M.j. LilleyAbstract:The Hammerhead Ribozyme undergoes a well-defined two-stage folding process induced by the sequential binding of two magnesium ions. These probably correspond to the formation of domain 2 (0-500 microM magnesium ions) and domain 1 (1-20 mM magnesium ions), respectively. In this study we have used fluorescence resonance energy transfer (FRET) to analyze the ion-induced folding of a number of variants of the Hammerhead Ribozyme. We find that both A14G and G8U mutations are highly destabilizing, such that these species are essentially unfolded under all conditions. Thus they appear to be blocked in the first stage of the folding process, and using uranyl-induced photocleavage we show that the core is completely accessible to this probe under these conditions. Changes at G5 do not affect the first transition but appear to provide a blockage at the second stage of folding; this is true of changes in the sugar (removal of the 2'-hydroxyl group) and base (G5C mutation, previously studied by comparative gel electrophoresis). Arrest of folding at this intermediate stage leads to a pattern of uranyl-induced photocleavage that is changed from the wild-type, but suggests a structure less open than the A14G mutant. Specific photocleavage at G5 is found only in the wild-type sequence, suggesting that this ion-binding site is formed late in the folding process. In addition to folding that is blocked at selected stages, we have also observed misfolding. Thus the A13G mutation appears to result in the ion-induced formation of a novel tertiary structure.
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Ion‐induced folding of the Hammerhead Ribozyme: a fluorescence resonance energy transfer study
The EMBO journal, 1997Co-Authors: Gurminder S. Bassi, Alastair I.h. Murchie, Frank Walter, Robert M. Clegg, David M.j. LilleyAbstract:The ion‐induced folding transitions of the Hammerhead Ribozyme have been analysed by fluorescence resonance energy transfer. The Hammerhead Ribozyme may be regarded as a special example of a three‐way RNA junction, the global structure of which has been studied by comparing the distances (as energy transfer efficiencies) between the ends of pairs of labelled arms for the three possible end‐to‐end vectors as a function of magnesium ion concentration. The data support two sequential ion‐dependent transitions, which can be interpreted in the light of the crystal structures of the Hammerhead Ribozyme. The first transition corresponds to the formation of a coaxial stacking between helices II and III; the data can be fully explained by a model in which the transition is induced by a single magnesium ion which binds with an apparent association constant of 8000–10 000 M −1 . The second structural transition corresponds to the formation of the catalytic domain of the Ribozyme, induced by a single magnesium ion with an apparent association constant of ∼1100 M −1 . The Hammerhead Ribozyme provides a well‐defined example of ion‐dependent folding in RNA.
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ion induced folding of the Hammerhead Ribozyme a fluorescence resonance energy transfer study
The EMBO Journal, 1997Co-Authors: Gurminder S. Bassi, Alastair I.h. Murchie, Frank Walter, Robert M. Clegg, David M.j. LilleyAbstract:The ion‐induced folding transitions of the Hammerhead Ribozyme have been analysed by fluorescence resonance energy transfer. The Hammerhead Ribozyme may be regarded as a special example of a three‐way RNA junction, the global structure of which has been studied by comparing the distances (as energy transfer efficiencies) between the ends of pairs of labelled arms for the three possible end‐to‐end vectors as a function of magnesium ion concentration. The data support two sequential ion‐dependent transitions, which can be interpreted in the light of the crystal structures of the Hammerhead Ribozyme. The first transition corresponds to the formation of a coaxial stacking between helices II and III; the data can be fully explained by a model in which the transition is induced by a single magnesium ion which binds with an apparent association constant of 8000–10 000 M −1 . The second structural transition corresponds to the formation of the catalytic domain of the Ribozyme, induced by a single magnesium ion with an apparent association constant of ∼1100 M −1 . The Hammerhead Ribozyme provides a well‐defined example of ion‐dependent folding in RNA.
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ionic interactions and the global conformations of the Hammerhead Ribozyme
Nature Structural & Molecular Biology, 1995Co-Authors: Gurminder S. Bassi, Alastair I.h. Murchie, Niels Erik Mollegard, Eberhard Von Kitzing, David M.j. LilleyAbstract:Here we investigate the global conformation of the Hammerhead Ribozyme. Electrophoretic studies demonstrate that the structure is folded in response to the concentration and type of ions present. Folding based on colinear alignment of arms II and III is suggested, with a variable angle subtended by the remaining helix I. In the probable active conformation, a small angle is subtended between helices I and II. Using uranyl photocleavage, an ion binding site has been detected in the long single-stranded region. The folded conformation could generate a pre-activation of the scissile bond to permit in-line attack of the 2′- hydroxyl group, with a bound metal ion playing an integral role in the chemistry.
Olke C. Uhlenbeck - One of the best experts on this subject based on the ideXlab platform.
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the structure function dilemma of the Hammerhead Ribozyme
Annual Review of Biophysics and Biomolecular Structure, 2005Co-Authors: Kenneth F Blount, Olke C. UhlenbeckAbstract:A powerful approach to understanding protein enzyme catalysis is to examine the structural context of essential amino acid side chains whose deletion or modification negatively impacts catalysis. In principle, this approach can be even more powerful for RNA enzymes, given the wide variety and subtlety of functionally modified nucleotides now available. Numerous recent success stories confirm the utility of this approach to understanding Ribozyme function. An anomaly, however, is the Hammerhead Ribozyme, for which the structural and functional data do not agree well, preventing a unifying view of its catalytic mechanism from emerging. To delineate the Hammerhead structure-function comparison, we have evaluated and distilled the large body of biochemical data into a consensus set of functional groups unambiguously required for Hammerhead catalysis. By examining the context of these functional groups within available structures, we have established a concise set of disagreements between the structural and functional data. The number and relative distribution of these inconsistencies throughout the Hammerhead reaffirms that an extensive conformational rearrangement from the fold observed in the crystal structure must be necessary for cleavage to occur. The nature and energetic driving force of this conformational isomerization are discussed.
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probing the binding of tb iii and eu iii to the Hammerhead Ribozyme using luminescence spectroscopy
Chemistry & Biology, 1999Co-Authors: Andrew L. Feig, Mark Panek, William Dew Horrocks, Olke C. UhlenbeckAbstract:Abstract Background: Divalent metal ions serve as structural as well as catalytic cofactors in the Hammerhead Ribozyme reaction. The natural cofactor in these reactions is Mg(II), but its spectroscopic silence makes it difficult to study. We previously showed that a single Tb(III) ion inhibits the Hammerhead Ribozyme by site-specific competition for a Mg(II) ion and therefore can be used as a spectroscopic probe for the Mg(II) it replaces. Results: Lanthanide luminescence spectroscopy was used to study the coordination environment around Tb(III) and Eu(III) ions bound to the structurally well-characterized site on the Hammerhead Ribozyme. Sensitized emission and direct excitation experiments show that a single lanthanide ion binds to the Ribozyme under these conditions and that three waters of hydration are displaced from the Tb(III) upon binding the RNA. Furthermore, we show that these techniques allow the comparison of binding affinities for a series of ions to this site. The binding affinities for ions at the G5 site correlates linearly with the function Z 2 /r of the aqua ion (where Z is the charge and r is the radius of the ion). Conclusions: This study compares the crystallographic nature of the G5 metal-binding site with solution measurements and gives a clearer picture of the coordination environment of this ion. These results provide one of the best characterized metal-binding sites from a Ribozyme, so we use this information to compare the RNA site with that of typical metalloproteins.
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Structural Variation Induced by Different Nucleotides at the Cleavage Site of the Hammerhead Ribozyme
Biochemistry, 1998Co-Authors: Jean-pierre Simorre, Lara Maloney, Nassim Usman, Leonid Beigelman, Olke C. Uhlenbeck, Pascale Legault, Narayan Baidya, Francine E. Wincott, Arthur PardiAbstract:The Hammerhead Ribozyme is capable of cleaving RNA substrates at 5' UX 3' sequences (where the cleavage site, X, can be A, C, or U). Hammerhead complexes containing dC, dA, dI, or rG nucleotides at the cleavage site have been studied by NMR. The rG at the cleavage site forms a Watson-Crick base pair with C3 in the conserved core of the Hammerhead, indicating that rG substrates inhibit the cleavage reaction by stabilizing an inactive conformation of the molecule. Isotope-edited NMR experiments on the Hammerhead complexes show that there are different short proton-proton distances between neighboring residues depending upon whether there is a dC or dA at the cleavage site. These NMR data demonstrate that there are significant differences in the structure and/or dynamics of the active-site residues in these Hammerhead complexes. Molecular dynamics calculations were used to model the conformations of the cleavage-site variants consistent with the NMR data. The solution conformations of the Hammerhead Ribozyme-substrate complexes are compared with the X-ray structure of the Hammerhead Ribozyme and are used to help understand the thermodynamic and kinetic differences among the cleavage-site variants.
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Hammerhead Ribozyme kinetics.
RNA (New York N.Y.), 1998Co-Authors: Tracy K. Stage-zimmermann, Olke C. UhlenbeckAbstract:The Hammerhead Ribozyme is a small RNA motif that self cleaves at a specific phosphodiester bond to produce 2′,3′ cyclic phosphate and 5′ hydroxyl termini (Hutchins et al., 1986; Forster & Symons, 1987a). The secondary structure of the Hammerhead consists of three helices of arbitrary sequence and length (designated I, II, and III) that intersect at 15 nucleotides termed the catalytic core (Fig. 1A) (Forster & Symons, 1987b; Hertel et al., 1992). The X-ray crystal structures of two Hammerhead Ribozyme–inhibitor complexes revealed that the core residues fold into two separate domains and the helices are arranged in a Y-shape conformation with helix I and helix II forming the upper portion of the Y (Pley et al., 1994; Scott et al., 1995). Although the Hammerhead is found as an intramolecular motif embedded in several RNAs in vivo (Symons, 1989), it can be assembled from two separate oligonucleotides (Fig. 1B) in three different arrangements (Uhlenbeck, 1987; Haseloff & Gerlach, 1988; Koizumi et al., 1988; Jeffries & Symons, 1989). In these bimolecular formats, the Hammerhead effects RNA cleavage in a similar manner to a true “enzyme,” proceeding through multiple rounds of substrate binding, cleavage, and product release (Uhlenbeck, 1987).
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Cryoenzymology of the Hammerhead Ribozyme.
RNA (New York N.Y.), 1998Co-Authors: Andrew L. Feig, Glenyss E. Ammons, Olke C. UhlenbeckAbstract:The technique of cryoenzymology has been applied to the Hammerhead Ribozyme in an attempt to uncover a structural rearrangement step prior to cleavage. Several cryosolvents were tested and 40% (v/v) methanol in water was found to perturb the system only minimally. This solvent allowed the measurement of Ribozyme activity between 30 and ‐33 8C. Eyring plots are linear down to ‐27 8C, but a drastic reduction in activity occurs below this temperature. However, even at extremely low temperatures, the rate is still quite pH dependent, suggesting that the chemical step rather than a structural rearrangement is still rate-limiting. The nonlinearity of the Eyring plot may be the result of a transition to a cold-denatured state or a glassed state.
Arthur Pardi - One of the best experts on this subject based on the ideXlab platform.
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Metal ion specificities for folding and cleavage activity in the Schistosoma Hammerhead Ribozyme.
RNA (New York N.Y.), 2008Co-Authors: Jennifer L. Boots, Marella D. Canny, Ehsan Azimi, Arthur PardiAbstract:The effects of various metal ions on cleavage activity and global folding have been studied in the extended Schistosoma Hammerhead Ribozyme. Fluorescence resonance energy transfer was used to probe global folding as a function of various monovalent and divalent metal ions in this Ribozyme. The divalent metals ions Ca2+, Mg2+, Mn2+, and Sr2+ have a relatively small variation (less than sixfold) in their ability to globally fold the Hammerhead Ribozyme, which contrasts with the very large difference (>10,000-fold) in apparent rate constants for cleavage for these divalent metal ions in single-turnover kinetic experiments. There is still a very large range (>4600-fold) in the apparent rate constants for cleavage for these divalent metal ions measured in high salt (2 M NaCl) conditions where the Ribozyme is globally folded. These results demonstrate that the identity of the divalent metal ion has little effect on global folding of the Schistosoma Hammerhead Ribozyme, whereas it has a very large effect on the cleavage kinetics. Mechanisms by which the identity of the divalent metal ion can have such a large effect on cleavage activity in the Schistosoma Hammerhead Ribozyme are discussed.
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Efficient ligation of the Schistosoma Hammerhead Ribozyme.
Biochemistry, 2007Co-Authors: Marella D. Canny, Fiona M. Jucker, Arthur PardiAbstract:The Hammerhead Ribozyme from Schistosoma mansoni is the best characterized of the natural Hammerhead Ribozymes. Biophysical, biochemical, and structural studies have shown that the formation of the loop−loop tertiary interaction between stems I and II alters the global folding, cleavage kinetics, and conformation of the catalytic core of this Hammerhead, leading to a Ribozyme that is readily cleaved under physiological conditions. This study investigates the ligation kinetics and the internal equilibrium between cleavage and ligation for the Schistosoma Hammerhead. Single turnover kinetic studies on a construct where the Ribozyme cleaves and ligates substrate(s) in trans showed up to 23% ligation when starting from fully cleaved products. This was achieved by an ∼2000-fold increase in the rate of ligation compared to a minimal Hammerhead without the loop−loop tertiary interaction, yielding an internal equilibrium that ranges from 2 to 3 at physiological Mg2+ ion concentrations (0.1−1 mM). Thus, the natura...
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Fast cleavage kinetics of a natural Hammerhead Ribozyme.
Journal of the American Chemical Society, 2004Co-Authors: Marella D. Canny, Fiona M. Jucker, Elizabeth A. Kellogg, Anastasia Khvorova, Sumedha Jayasena, Arthur PardiAbstract:The Hammerhead Ribozyme is a small RNA motif that catalyzes the cleavage and ligation of RNA. The well-studied minimal Hammerhead motif is inactive under physiological conditions and requires high Mg2+ concentrations for efficient cleavage. In contrast, natural Hammerheads are active under physiological conditions and contain motifs outside the catalytic core that lower the requirement for Mg2+. Single-turnover kinetics were used here to characterize the Mg2+ and pH dependence for cleavage of a trans-cleaving construct of the Schistosoma mansoni natural Hammerhead Ribozyme. Compared to the minimal Hammerhead motif, the natural Schistosoma Ribozyme requires 100-fold less Mg2+ to achieve a cleavage rate of 1 min-1. The improved catalysis results from tertiary interactions between loops in stems I and II and likely arises from increasing the population of the active conformation. Under optimum pH and Mg2+ conditions this Ribozyme cleaves at over 870 min-1 at 25 °C, further demonstrating the impressive cataly...
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the global conformation of the Hammerhead Ribozyme determined using residual dipolar couplings
Biochemistry, 2002Co-Authors: Kent Bondensgaard, Emilia T Mollova, Arthur PardiAbstract:The global structure of the Hammerhead Ribozyme was determined in the absence of Mg(2+) by solution NMR experiments. The Hammerhead Ribozyme motif forms a branched structure consisting of three helical stems connected to a catalytic core. The (1)H-(15)N and (1)H-(13)C residual dipolar couplings were measured in a set of differentially (15)N/(13)C-labeled Ribozymes complexed with an unlabeled noncleavable substrate. The residual dipolar couplings provide orientation information on both the local and the global structure of the molecule. Analysis of the residual dipolar couplings demonstrated that the local structure of the three helical stems in solution is well modeled by an A-form conformation. However, the global structure of the Hammerhead in solution in the absence of Mg(2+) is not consistent with the Y-shaped conformation observed in crystal structures of the Hammerhead. The residual dipolar couplings for the helical stems were combined with standard NOE and J coupling constant NMR data from the catalytic core. The NOE data show formation of sheared G-A base pairs in domain 2. These NMR data were used to determine the global orientation of the three helical stems in the Hammerhead. The Hammerhead forms a rather extended structure under these conditions with a large angle between stems I and II ( approximately 153 degrees ), a smaller angle between stems II and III ( approximately 100 degrees ), and the smallest angle between stems I and III ( approximately 77 degrees ). The residual dipolar coupling data also contain information on the dynamics of the molecule and were used here to provide qualitative information on the flexibility of the helical domains in the Hammerhead Ribozyme-substrate complex.
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Identification and characterization of a novel high affinity metal-binding site in the Hammerhead Ribozyme.
RNA (New York N.Y.), 1999Co-Authors: Mark R. Hansen, Leonid Beigelman, Jean-pierre Simorre, Paul Hanson, Victor Mokler, Laurent Bellon, Arthur PardiAbstract:A novel metal-binding site has been identified in the Hammerhead Ribozyme by 31 P NMR. The metal-binding site is associated with the A13 phosphate in the catalytic core of the Hammerhead Ribozyme and is distinct from any previously identified metal-binding sites. 31 P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250‐570 m Ma t 25 8C for binding of a single Mg 21 ion. The NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the Hammerhead Ribozyme. These NMR data were combined with the X-ray structure of the Hammerhead Ribozyme (Pley HW, Flaherty KM, McKay DB. 1994. Nature 372:68‐74) to model RNA ligands involved in binding the metal at this A13 site. In this model, the A13 metal-binding site is structurally similar to the previously identified A9 metal-binding site and illustrates the symmetrical nature of the
