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1.
The hammerhead ribozyme undergoes a well-defined two-stage conformational folding process, induced by the binding of magnesium ions. In this study, we have used isothermal titration calorimetry to analyze the thermodynamics of magnesium binding and magnesium ion-induced folding of the ribozyme. Binding to the natural sequence ribozyme is strongly exothermic and can be analyzed in terms of sequential interaction at two sites with association constants K(A) = 480 and 2840 M(-1). Sequence variants of the hammerhead RNA give very different isothermal titration curves. An A14G variant that cannot undergo ion-induced folding exhibits endothermic binding. By contrast, a deoxyribose G5 variant that can undergo only the first of the two folding transitions gives a complex titration curve. However, despite these differences the ITC data for all three species can be analyzed in terms of the sequential binding of magnesium ions at two sites. While the binding affinities are all in the region of 10(3) M(-1), corresponding to free energies of Delta G degrees = -3.5 to -4 kcal mol(-1), the enthalpic and entropic contributions show much greater variation. The ITC experiments are in good agreement with earlier conformational studies of the folding of the ion-induced folding of the hammerhead ribozyme. 相似文献
2.
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. 相似文献
3.
Calreticulin (CRT) is a soluble, lectin chaperone found in the endoplasmic reticulum of eukaryotes. It binds the N-glycosylated
polypeptides via the glycan intermediate Glc1Man5–9GlcNAc2, present on the target glycoproteins. Earlier we have studied interactions of substrate with CRT by isothermal titration
calorimetry (ITC) and molecular modeling, to establish that CRT recognizes the Glcα1–3 linkage and forms contacts with each
saccharide moiety of the oligosaccharide Glcα1–3Manα1–2Manα1–2Man. We also delineated the amino acid residues in the sugar
binding pocket of CRT that play a crucial role in sugar–CRT binding. Here, we have used mono-deoxy analogues of the trisaccharide
unit Glcα1–3Manα1–2Man to determine the role of various hydroxyl groups of the sugar substrate in sugar–CRT interactions.
Using the thermodynamic data obtained by ITC with these analogues we demonstrate that the 3-OH group of Glc1 plays an important
role in sugar–CRT binding, whereas the 6-OH group does not. Also, the 4-OH, 6-OH of Man2 and 3-OH, 4-OH of Man3 in the trisaccharide
are involved in binding, of which 6-OH of Man2 and 4-OH of Man3 have a more significant role to play. This study sheds light
further on the interactions between the substrate sugar of glycoproteins and the lectin chaperone CRT. 相似文献
4.
Heat capacity changes in RNA folding: application of perturbation theory to hammerhead ribozyme cold denaturation
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In proteins, empirical correlations have shown that changes in heat capacity (ΔCP) scale linearly with the hydrophobic surface area buried upon folding. The influence of ΔCP on RNA folding has been widely overlooked and is poorly understood. In addition to considerations of solvent reorganization, electrostatic effects might contribute to ΔCPs of folding in polyanionic species such as RNAs. Here, we employ a perturbation method based on electrostatic theory to probe the hot and cold denaturation behavior of the hammerhead ribozyme. This treatment avoids much of the error associated with imposing two-state folding models on non-two-state systems. Ribozyme stability is perturbed across a matrix of solvent conditions by varying the concentration of NaCl and methanol co-solvent. Temperature-dependent unfolding is then monitored by circular dichroism spectroscopy. The resulting array of unfolding transitions can be used to calculate a ΔCP of folding that accurately predicts the observed cold denaturation temperature. We confirm the accuracy of the calculated ΔCP by using isothermal titration calorimetry, and also demonstrate a methanol-dependence of the ΔCP. We weigh the strengths and limitations of this method for determining ΔCP values. Finally, we discuss the data in light of the physical origins of the ΔCPs for RNA folding and consider their impact on biological function. 相似文献
5.
The hammerhead ribozyme, a small catalytic RNA molecule, cleaves, in the presence of magnesium ions, a specific phosphodiester bond within its own backbone, leading to 23-cyclic phosphate and 5-OH extremities. In order to study the dynamical flexibility of the hammerhead RNA, we performed molecular dynamics simulations of the solvated crystal structure of an active hammerhead ribozyme, obtained after flash-freezing crystals soaked with magnesium. Because of a careful equilibration protocol and the use of the Ewald summation in calculating the electrostatic interactions, the RNA structure remained close to the crystal structure, as attested by a root-mean-square deviation below 2.5 A after 750 ps of simulation. All Watson-Crick base pairs were intact at the end of the simulations. The tertiary interactions, such as the sheared G.A pairs and the U-turn, important for the stabilisation of the three-dimensional RNA fold, were also retained. The results demonstrate that molecular dynamics simulations can be successfully used to investigate the dynamical behaviour of a ribozyme, thus, opening a road to study the role of transient structural changes involved in ribozyme catalysis. 相似文献
6.
A helical twist-induced conformational switch activates cleavage in the hammerhead ribozyme 总被引:6,自引:0,他引:6
We have captured the structure of a pre-catalytic conformational intermediate of the hammerhead ribozyme using a phosphodiester tether formed between I and Stem II. This phosphodiester tether appears to mimic interactions in the wild-type hammerhead RNA that enable switching between nuclease and ligase activities, both of which are required in the replicative cycles of the satellite RNA viruses from which the hammerhead ribozyme is derived. The structure of this conformational intermediate reveals how the attacking nucleophile is positioned prior to cleavage, and demonstrates how restricting the ability of Stem I to rotate about its helical axis, via interactions with Stem II, can inhibit cleavage. Analogous covalent crosslinking experiments have demonstrated that imposing such restrictions on interhelical movement can change the hammerhead ribozyme from a nuclease to a ligase. Taken together, these results permit us to suggest that switching between ligase and nuclease activity is determined by the helical orientation of Stem I relative to Stem II. 相似文献
7.
Electron paramagnetic resonance (EPR) spectroscopy was used to examine changes in internal structure and dynamics of the hammerhead ribozyme upon metal ion induced folding, changes in pH, and the presence and absence of ribozyme inhibitors. A nitroxide spin-label was attached to nucleotide U7 of the HH16 catalytic core, and this modified ribozyme was observed to retain catalytic activity. U7 was shown by EPR spectroscopy to be more mobile in the ribozyme-product complex than in either the unfolded ribozyme or the ribozyme-substrate complex. A two-step divalent metal ion dependent folding pathway was observed for the ribozyme-substrate complex with a weak first transition observed at 0.25 mM Mg2+ and a strong second transition observed around 10 mM Mg2+, in agreement with studies using other biophysical and biochemical techniques. Previously, ribozyme activity was observed in the absence of divalent metal ions and the presence of high concentrations of monovalent metal ions, although the activity was less than that observed in the presence of divalent metal ions. Here, we observed similar dynamics for U7 in the presence of 4 M Na+ or Li+, which were distinctively different than that observed in the presence of 10 mM Mg2+, indicating that U7 of the catalytic core forms a different microenvironment under monovalent versus divalent metal ion conditions. Interestingly, the catalytically efficient microenvironment of U7 was similar to that observed in a solution containing 1 M Na+ upon addition of one divalent metal ion per ribozyme. In summary, these results demonstrate that changes in local dynamics, as detected by EPR spectroscopy, can be used to study conformational changes associated with RNA folding and function. 相似文献
8.
Ion-induced folding of the hammerhead ribozyme: a fluorescence resonance energy transfer study. 总被引:8,自引:1,他引:7
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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 approximately 1100 M-1. The hammerhead ribozyme provides a well-defined example of ion-dependent folding in RNA. 相似文献
9.
Probing RNA tertiary structure: interhelical crosslinking of the hammerhead ribozyme. 总被引:3,自引:0,他引:3
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Distinct structural models for the hammerhead ribozyme derived from single-crystal X-ray diffraction and fluorescence resonance energy transfer (FRET) measurements have been compared. Both models predict the same overall geometry, a wishbone shape with helices II and III nearly colinear and helix I positioned close to helix II. However, the relative orientations of helices I and II are different. To establish whether one of the models represents a kinetically active structure, a new crosslinking procedure was developed in which helices I and II of hammerhead ribozymes were disulfide-crosslinked via the 2' positions of specific sugar residues. Crosslinking residues on helices I and II that are close according to the X-ray structure did not appreciably reduce the catalytic efficiency. In contrast, crosslinking residues closely situated according to the FRET model dramatically reduced the cleavage rate by at least three orders of magnitude. These correlations between catalytic efficiencies and spatial proximities are consistent with the X-ray structure. 相似文献
10.
Structure, folding and activity of the VS ribozyme: importance of the 2-3-6 helical junction 总被引:1,自引:0,他引:1
The VS nucleolytic ribozyme has a core comprising five helices organized by two three-way junctions. The ribozyme can act in trans on a hairpin-loop substrate, with which it interacts via tertiary contacts. We have determined that one of the junctions (2-3-6) undergoes two-stage ion-dependent folding into a stable conformation, and have determined the global structure of the folded junction using long-range distance restraints derived from fluorescence resonance energy transfer. A number of sequence variants in the junction are severely impaired in ribozyme cleavage, and there is good correlation between changes in activity and alteration in the folding of junction 2-3-6. These studies point to a special importance of G and A nucleotides immediately adjacent to helix II, and comparison with a similar junction of known structure indicates that this could adopt a guanine-wedge structure. We propose that the 2-3-6 junction organizes important aspects of the structure of the ribozyme to facilitate productive association with the substrate, and suggest that this results in an interaction between the substrate and the A730 loop to create the active complex. 相似文献
11.
The ion-induced folding of the hammerhead ribozyme: core sequence changes that perturb folding into the active conformation. 总被引:2,自引:1,他引:1
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The hammerhead ribozyme undergoes an ion-dependent folding process into the active conformation. We find that the folding can be blocked at specific stages by changes of sequence or functionality within the core. In the the absence of added metal ions, the global structure of the hammerhead is extended, with a large angle subtended between stems I and II. No core sequence changes appear to alter this geometry, consistent with an unstructured core under these conditions. Upon addition of low concentrations of magnesium ions, the hammerhead folds by an association of stems II and III, to include a large angle between them. This stage is inhibited or altered by mutations within the oligopurine sequence lying between stems II and III, and folding is completely prevented by an A14G mutation. Further increase in magnesium ion concentration brings about a second stage of folding in the natural sequence hammerhead, involving a reorientation of stem I, which rotates around into the same direction of stem II. Because this transition occurs over the same range of magnesium ion concentration over which the hammerhead ribozyme becomes active, it is likely that the final conformation is most closely related to the active form of the structure. Magnesium ion-dependent folding into this conformation is prevented by changes at G5, notably removal of the 2'-hydroxyl group and replacement of the base by cytidine. The ability to dissect the folding process by means of sequence changes suggests that two separate ion-dependent stages are involved in the folding of the hammerhead ribozyme into the active conformation. 相似文献
12.
Alpha-crystallin, composed of two subunits, alphaA and alphaB, has been shown to function as a molecular chaperone that prevents aggregation of other proteins under stress conditions. The exposed hydrophobic surfaces of alpha-crystallins have been implicated in this process, but their exact role has not been elucidated. In this study, we quantify the hydrophobic surfaces of alphaA- and alphaB-crystallins by isothermal titration calorimetry using 8-anilino-1-napthalenesulfonic acid (ANS) as a hydrophobic probe and analyze its correlation to the chaperone potential of alphaA- and alphaB-crystallins under various conditions. Two ANS binding sites, one with low and another with high affinity, were clearly detected, with alphaB showing a higher number of sites than alphaA at 30 degrees C. In agreement with the higher number of hydrophobic sites, alphaB-crystallin demonstrated higher chaperone activity than alphaA at this temperature. Thermodynamic analysis of ANS binding to alphaA- and alphaB-crystallins indicates that high affinity binding is driven by both enthalpy and entropy changes, with entropy dominating the low affinity binding. Interestingly, although the number of ANS binding sites was similar for alphaA and alphaB at 15 degrees C, alphaA was more potent than alphaB in preventing aggregation of the insulin B-chain. Although there was no change in the number of high affinity binding sites of alphaA and alphaB for ANS upon preheating, there was an increase in the number of low affinity sites of alphaA and alphaB. Preheated alphaA, in contrast to alphaB, exhibited remarkably enhanced chaperone activity. Our results indicate that although hydrophobicity appears to be a factor in determining the chaperone-like activity of alpha-crystallins, it does not quantitatively correlate with the chaperone function of alpha-crystallins. 相似文献
13.
The hammerhead ribozyme is a more efficient ribonuclease than an RNA ligase. Under typical reaction conditions, the rate of RNA chain cleavage is approximately 100-fold faster than the rate of the reverse ligation reaction such that virtually all of the hammerhead is in its cleaved form at equilibrium. Here we show that the introduction of a crosslink away from the catalytic core of the hammerhead has little effect on the cleavage rate but dramatically increases the ligation rate, thereby making the hammerhead an efficient RNA ligase. This experiment emphasizes the role of molecular flexibility in defining the properties of a macromolecular catalyst and suggests why other small ribozymes are more efficient ligases than ribonucleases. 相似文献
14.
15.
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. 相似文献
16.
The catalytic activity of the hammerhead ribozyme is limited by its ability to fold into the native tertiary structure. Analysis of folding has been hampered by a lack of assays that can independently monitor the environment of nucleobases throughout the ribozyme-substrate complex in real time. Here, we report the development and application of a new folding assay in which we use pyrrolo-cytosine (pyC) fluorescence to (1) probe active-site formation, (2) examine the ability of peripheral ribozyme domains to support native folding, (3) identify a pH-dependent conformational change within the ribozyme, and (4) explore its influence on the equilibrium between the folded and unfolded core of the hammerhead ribozyme. We conclude that the natural ribozyme folds in two distinct noncooperative steps and the pH-dependent correlation between core folding and activity is linked to formation of the G8-C3 base pair. 相似文献
17.
Ivan Dotu Juan Antonio Garcia-Martin Betty L. Slinger Vinodh Mechery Michelle M. Meyer Peter Clote 《Nucleic acids research》2014,42(18):11752-11762
Nanotechnology and synthetic biology currently constitute one of the most innovative, interdisciplinary fields of research, poised to radically transform society in the 21st century. This paper concerns the synthetic design of ribonucleic acid molecules, using our recent algorithm, RNAiFold, which can determine all RNA sequences whose minimum free energy secondary structure is a user-specified target structure. Using RNAiFold, we design ten cis-cleaving hammerhead ribozymes, all of which are shown to be functional by a cleavage assay. We additionally use RNAiFold to design a functional cis-cleaving hammerhead as a modular unit of a synthetic larger RNA. Analysis of kinetics on this small set of hammerheads suggests that cleavage rate of computationally designed ribozymes may be correlated with positional entropy, ensemble defect, structural flexibility/rigidity and related measures. Artificial ribozymes have been designed in the past either manually or by SELEX (Systematic Evolution of Ligands by Exponential Enrichment); however, this appears to be the first purely computational design and experimental validation of novel functional ribozymes. RNAiFold is available at http://bioinformatics.bc.edu/clotelab/RNAiFold/. 相似文献
18.
In its natural context, the hairpin ribozyme is constructed around a four-way helical junction. This presents the two loops that interact to form the active site on adjacent arms, requiring rotation into an antiparallel structure to bring them into proximity. In the present study we have compared the folding of this form of the ribozyme and subspecies lacking either the loops or the helical junction using fluorescence resonance energy transfer. The complete ribozyme as a four-way junction folds into an antiparallel structure by the cooperative binding of magnesium ions, requiring 20-40 microM for half-maximal extent of folding ([Mg2+]1/2) and a Hill coefficient n = 2. The isolated junction (lacking the loops) also folds into a corresponding antiparallel structure, but does so noncooperatively (n = 1) at a higher magnesium ion concentration ([Mg2+]1/2 = 3 mM). Introduction of a G + 1A mutation into loop A of the ribozyme results in a species with very similar folding to the simple junction, and complete loss of ribozyme activity. Removal of the junction from the ribozyme, replacing it either with a strand break (serving as a hinge) or a GC5 bulge, results in greatly impaired folding, with [Mg2+]1/2 > 20 mM. The results indicate that the natural form of the ribozyme undergoes ion-induced folding by the cooperative formation of an antiparallel junction and loop-loop interaction to generate the active form of the ribozyme. The four-way junction thus provides a scaffold in the natural RNA that facilitates the folding of the ribozyme into the active form. 相似文献
19.
D B McKay 《RNA (New York, N.Y.)》1996,2(5):395-403
20.
Mixed DNA/RNA polymers are cleaved by the hammerhead ribozyme. 总被引:3,自引:0,他引:3
A series of chemically synthesized oligodeoxyribonucleotides containing one or two ribonucleotides (DNA/RNA mixed polymers) at and/or adjacent to the cleavage site of the substrate can be cleaved by the "hammerhead" ribozyme. In comparison with the all-RNA substrate, the predominantly deoxyribonucleotide substrates have (1) lower optimal temperatures of cleavage, (2) approximately 6-fold higher Km's and 7-fold lower kcat's at 30 degrees C, and (3) 15-fold higher Km's and 8-fold lower kcat's at 37 degrees C. The extent to which the RNA substrate cleavage is inhibited in the presence of an all-DNA (KI = 13 microM) and an RNA substrate analogue with a dC at the cleavage site (KI = 0.96 microM) supports the contention that the formation of the ribozyme-substrate complex with the predominantly deoxyribonucleotide substrates (D substrates) is impaired. The weaker binding of D substrates was confirmed by thermal denaturation and determination of the Tm of the complex. Analysis of the kinetic data also suggests that the conformation of the catalytic core of the ribozyme-substrate complex differs from that of the all-RNA complex, a change that results from the presence of a DNA/RNA heteroduplex in the complex. 相似文献