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A I Murchie

Publications and source records attributed to A I Murchie.

At least 19 recordsLinked to original sources
PubMed1999

RNA folding and misfolding of the hammerhead ribozyme.

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.

Adenine↗
PubMed1998

Folding of the four-way RNA junction of the hairpin ribozyme.

The hairpin ribozyme consists of two loop-carrying duplexes (called A and B) that are adjacent arms of a four-way junction in its natural context in the viral RNA. We have shown previously that the activity of the ribozyme is strongly influenced by the structure adopted by the junction. In this study, we have used fluorescence resonance energy transfer to analyze the conformation and folding of the isolated four-way junction. Like other four-way RNA junctions, in the absence of added metal ions this junction adopts a square configuration of coaxially stacked arms, based on A on D and B on C stacking. Upon addition of magnesium ions, the junction undergoes an ion-induced transition to an antiparallel conformation. The data are consistent with folding induced by the binding of a single ion, with an apparent association constant in the range of 2000 M-1. Other divalent metal ions (calcium or manganese) can also induce this change in structure; however, sodium ions are unable to substitute for these ions, and are slightly inhibitory with respect to the transition. The loop-free hairpin junction adopts the same stacking conformer as the full ribozyme, but forms a more symmetrical X-shaped structure. In addition, the apparent stoichiometry of structural ion binding is lower for the isolated junction, and the affinity is considerably lower.

Calcium↗
PubMed1998

Structure and activity of the hairpin ribozyme in its natural junction conformation: effect of metal ions.

The natural form of the hairpin ribozyme consists of a four-way RNA junction of which the single-stranded loop-carrying helices are adjacent arms. The junction can be regarded as providing a framework for constructing the active ribozyme, and the rate of cleavage can be modulated by changing the conformation of the junction. We find that the junction-based form of the hairpin ribozyme is active in magnesium, calcium, or strontium ions, but not in manganese, cadmium, or sodium ions. Using fluorescence resonance energy transfer experiments, we have investigated the global structure of the ribozyme. The basic folding of the construct is based on pairwise helical stacking, so that the two loop-carrying arms are located on opposite stacked helical pairs. In the presence of magnesium, calcium, or strontium ions, the junction of the ribozyme undergoes a rotation into a distorted antiparallel geometry, creating close physical contact between the two loops. Manganese ions induce the same global folding, but no catalytic activity; this change in global conformation is therefore necessary but not sufficient for catalytic activity. Fitting the dependence of the conformation on ionic concentration to a two-state model suggests that cooperative binding of two ions is required to bring about the folding. However, further ion binding is required for cleavage activity. Cobalt hexammine ions also bring about global folding, while spermidine generates a more symmetrical form of the antiparallel structure. Cadmium ions generate a different folded form, interpreted in terms of close loop-loop association while the junction is unfolded. Sodium ions were unable to induce any folding of the ribozyme, which remained slightly parallel. These results are consistent with a folding process induced by the binding of two group IIA metal ions, distributed between the junction and the loop interface.

Base Sequence↗
PubMed1998

Exchange between stacking conformers in a four-Way DNA junction.

Four-way DNA junctions undergo metal ion-induced folding by means of pairwise coaxial stacking of helical arms in one of two possible conformers that depend on the choice of stacking partners. For most such junctions there is a significant bias toward one conformer over the other. In this study we have characterized a four-way DNA junction in which there is rapid exchange between equal populations of the two possible stacking conformers. Analysis of the global conformation using comparative gel electrophoresis gives results consistent with either a tetrahedral disposition of the four arms or an equilibrium between equal populations of the two alternative stacked X-structures. Protection of bases at the center of the junction against attack by osmium tetroxide indicates that base stacking is preserved through the point of strand exchange. Cleavage across the point of strand exchange by the restriction enzyme MboII is consistent with pairwise coaxial stacking of helical arms. Taken together, these indicate that the junction adopts the stacked X-structure, but unusually there appears to be little bias for one stacking conformer over the other. Complete digestion of junctions by MboII demonstrates that all the molecules in solution pass through a given conformer during the time of incubation, demonstrating that exchange between conformers must occur. This is true even for minor stacking conformers in strongly biased junctions. Comparative gel electrophoresis shows that sequence changes at the third position out from the point of strand exchange can have a marked influence on the relative stability of the stacking conformers.

Cations, Divalent↗
PubMed1998

Folding of the hairpin ribozyme in its natural conformation achieves close physical proximity of the loops.

The hairpin ribozyme is a self-cleaving motif found in the negatives strand of the satellite RNA of some plant viruses. In its natural context, the ribozyme comprises four helices, two of which contain conserved formally unpaired loops, that are adjacent arms of a four-way RNA junction. We show that the arms that would carry these loops are brought close together in the global conformation of the isolated junction. Using fluorescence resonance energy transfer, we demonstrate a two-magnesium ion-dependent conformational transition of the complete ribozyme that brings the loopbearing arms into close physical proximity. The ribozyme is active as a four-way junction, and the rate of cleavage may be modulated by the conformation of the four-way junction.

Base Sequence↗
PubMed1998

Global structure of four-way RNA junctions studied using fluorescence resonance energy transfer.

Four-way helical junctions are found widely in natural RNA species. In this study, we have studied the conformation of two junctions by fluorescence resonance energy transfer. We show that the junctions are folded by pairwise coaxial helical stacking, forming one predominant stacking conformer in both examples studied. At low magnesium ion concentrations, the helical axes of both junctions are approximately perpendicular. One junction undergoes a rotation into a distorted antiparallel structure induced by the binding of a single magnesium ion. By contrast, the axes of the four-way junction of the U1 snRNA remain approximately perpendicular under all conditions examined, and we have determined the stacking conformer adopted.

Base Sequence↗
PubMed1997

Ion-induced folding of the hammerhead ribozyme: a fluorescence resonance energy transfer study.

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.

Base Sequence↗
PubMed1997

Global structure of three-way DNA junctions with and without additional unpaired bases: a fluorescence resonance energy transfer analysis.

The structure of three-way DNA junctions with and without extrahelical adenine nucleotides in one strand at the branch point of the junction (i.e., An bulges with n = 0, 1, 2, and 3) has been investigated by fluorescence resonance energy transfer. The structure of the junction without bulged nucleotides was found to have a symmetric trigonal geometry. With bulges, the arrangement of the arms becomes asymmetrical. The energy transfer results suggest a model of bulged junctions where the angle between two of the arms is significantly smaller than between the other two pairs of arms. The acute angle becomes smaller as the number of nucleotides in the bulge increases. The FRET efficiencies of the junctions are the same in the presence of Mg++ and Na+ ions.

Base Composition↗
PubMed1997

Severe axial bending of RNA induced by the U1A binding element present in the 3' untranslated region of the U1A mRNA.

The 3' untranslated region of the U1A mRNA contains a binding site for the U1A protein that consists of two asymmetric internal bulges. The bulges each comprise a loop of seven unpaired bases opposing a single base (termed a U1A box). The seven-base loops are located on opposite strands, distributed in a symmetrical manner about the intervening four-base duplex. We have investigated the global conformation of this binding element. Comparison of electrophoretic mobilities of RNA duplexes interrupted by a single U1A box with a series of duplexes of the same length containing oligoadenine bulges indicates that the individual boxes cause a substantial kinking of the helix axis, estimated to be 90 (+/- 10) degrees. A series of RNA duplexes were constructed containing a U1A box separated from an A5 bulge by a duplex section of length between 3 and 21 bp. It was found that the electrophoretic mobilities of these species varied sinusoidally, indicating that the U1A box introduces a defined kink into the RNA helix, rather than a point of flexibility. Electrophoretic experiments with the complete U1A binding element suggest that the axial trajectories of the two U1A boxes combine to give an approximately in-line, 180 degrees change in duplex direction.

Base Composition↗
PubMed1997

The crystal structure of a parallel-stranded guanine tetraplex at 0.95 A resolution.

In both DNA and RNA, stretches of guanine bases can form stable four-stranded helices in the presence of sodium or potassium ions. Sequences with a propensity to form guanine tetraplexes have been found in chromosomal telomers, immunoglobulin switch regions, and recombination sites. We report the crystal structure at 0.95 A resolution of a parallel-stranded tetraplex formed by the hexanucleotide d(TG4T) in the presence of sodium ions. The four strands form a right-handed helix that is stabilized by hydrogen-bonding tetrads of co-planar guanine bases. Well-resolved sodium ions are found between and, at defined points, within tetrad planes and are coordinated with the guanine O6 groups. Nine calcium ions have been identified, each with a well-defined hepta-coordinate hydration shell. Hydrogen-bonding water patterns are observed within the tetraplex's helical grooves and clustered about the phosphate groups. Water molecules in the groove may form a hydrogen bond with the O4', and may affect the stacking behavior of guanine. Two distinct stacking arrangements are noted for the guanine tetrads. The thymine bases do not contribute to the four-stranded conformation, but instead stack to stabilize the crystal lattice. We present evidence that the sugar conformation is strained and propose that this originates from forces that optimize guanine base stacking. Discrete conformational disorder is observed at several places in the phosphodiester backbone, which results from a simple crankshaft rotation that requires no net change in the sugar conformation.

Base Composition↗
PubMed1997

Nucleic acid structure and recognition.

We review the global structures adopted by branched nucleic acids, including three- and four-way helical junctions in DNA and RNA. We find that some general folding principles emerge. First, all the structures exhibit a tendency to undergo pairwise coaxial helical stacking when permitted by the local stereochemistry of strand exchange. Second, metal ions generally play an important role in facilitating folding of branched nucleic acids. These principles can be applied to functionally important branched nucleic acids, such as the Holliday DNA junction of genetic recombination, and the hammerhead ribozyme in RNA.

Journal Article↗
PubMed1996

Human MutSalpha recognizes damaged DNA base pairs containing O6-methylguanine, O4-methylthymine, or the cisplatin-d(GpG) adduct.

Bacterial and mammalian mismatch repair systems have been implicated in the cellular response to certain types of DNA damage, and genetic defects in this pathway are known to confer resistance to the cytotoxic effects of DNA-methylating agents. Such observations suggest that in addition to their ability to recognize DNA base-pairing errors, members of the MutS family may also respond to genetic lesions produced by DNA damage. We show that the human mismatch recognition activity MutSalpha recognizes several types of DNA lesion including the 1,2-intrastrand d(GpG) crosslink produced by cis-diamminedichloroplatinum(II), as well as base pairs between O6-methylguanine and thymine or cytosine, or between O4-methylthymine and adenine. However, the protein fails to recognize 1,3-intrastrand adduct produced by trans-diamminedichloroplatinum(II) at a d(GpTpG) sequence. These observations imply direct involvement of the mismatch repair system in the cytotoxic effects of DNA-methylating agents and suggest that recognition of 1,2-intrastrand cis-diamminedichloroplatinum(II) adducts by MutSalpha may be involved in the cytotoxic action of this chemotherapeutic agent.

Adenosine Triphosphatases↗
PubMed1996

The ion-induced folding of the hammerhead ribozyme: core sequence changes that perturb folding into the active conformation.

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.

Base Sequence↗
PubMed1995

The global folding of four-way helical junctions in RNA, including that in U1 snRNA.

Helical junctions are important elements in the architecture of folded RNA molecules. The global geometry of fully base-paired four-way junctions between RNA helices has been analyzed by comparative gel electrophoresis. Junctions appear to fold by pairwise coaxial helical stacking in one of two possible stereochemically equivalent isomers based upon alternative selections of stacking partners. In the presence of 1 mM Mg2+, the two continuous helical axes are approximately at right angles to each other for all junctions studied, but the RNA junctions exhibit significant sequence-dependent differences in their structures as a function of ionic conditions. The four-way junction found in the U1 snRNA folded by coaxial helical stacking. It retained the 90 degrees crossed stacked structure under all ionic conditions tested, despite the presence of a G.A mismatch at the point of strand exchange.

Base Sequence↗
PubMed1995

Structure of the four-way DNA junction and its interaction with proteins.

The four-way DNA junction is an important intermediate in recombination processes; it is, the substrate for different enzyme activities. In solution, the junction adopts a right-handed, antiparallel-stacked X-structure formed by the pairwise coaxial-stacking of helical arms. The stereochemistry is determined by the juxtaposition of grooves and backbones, which is optimal when the smaller included angle is 60 degrees. The antiparallel structure has two distinct sides with major and minor groove-characteristics, respectively. The folding process requires the binding of metal cations, in the absence of which, the junction remains extended without helix-helix stacking. The geometry of the junction can be perturbed by the presence of certain base-base mispairs or phosphodiester discontinuities located at the point of strand exchange. The four-way DNA junction is selectively cleaved by a number of resolving enzymes. In a number of cases, these appear to recognize the minor groove face of the junction and are functionally divisible into activities that recognize and bind the junction, and a catalytic activity. Some possible mechanisms for the recognition of branched DNA structure are discussed.

DNA↗
PubMed1995

Ionic interactions and the global conformations of the hammerhead ribozyme.

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 preactivation 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.

Base Sequence↗
PubMed1994

Kinking of DNA and RNA helices by bulged nucleotides observed by fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) has been used to demonstrate the bending of DNA and RNA helices for three series of double-stranded molecules containing bulge loops of unopposed adenosine nucleotides (An, n = 0-9). Fluorescein and rhodamine were covalently attached to the 5' termini of the two component strands. Three different methods were applied to measure the FRET efficiencies. The extent of energy transfer within each series increases as the number of bulged nucleotides varies from 1 to 7, indicating a shortening of the end-to-end distance. This is consistent with a bending of DNA and RNA helices that is greater for larger bulges. The FRET efficiency for DNA molecules with A9 bulges is lower than the efficiency for the corresponding A7 bulged molecules, although the A9 molecules exhibit increased electrophoretic retardation. Ranges of bending angles can be estimated from the FRET results.

Adenine Nucleotides↗