The folding of the hairpin ribozyme: Dependence on the loops and the junction

Zhong, Zhao; Wilson, Timothy J.; Maxwell, Kaera; Lilley, David M.J.

doi:10.1017/s1355838200001230

Cited by 57 publications

(52 citation statements)

References 69 publications

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“…Deletions in helix 1 reduce substrate binding affinity, consistent with previous results (Donahue et al 2000). The finding that helix 1 is important for formation of secondary structure, but not tertiary structure, is consistent with the results of biophysical and crystallographic studies, which show that helices 1 and 4 approach one another but do not form functional contacts during tertiary structure formation Porschke et al 1999;Zhao et al 2000;Rupert and Ferré-D'Amaré 2001). The modest in- This complex cleaves at a rate only 2.5-fold lower than that of the wild-type construct.…”

Section: Discussionsupporting

confidence: 89%

Modifications and deletions of helices within the hairpin ribozyme–substrate complex: An active ribozyme lacking helix 1

Pinard

Lambert²,

Pothiawala³

et al. 2004

RNA

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Within the hairpin ribozyme, structural elements required for formation of the active tertiary structure are localized in two independently folding domains, each consisting of an internal loop flanked by helical elements. Here, we present results of a systematic examination of the relationship between the structure of the helical elements and the ability of the RNA to form the catalytically active tertiary structure. Deletions and mutational analyses indicate that helix 1 (H1) in domain A can be entirely eliminated, while segments of helices 2, 3, and 4 can also be deleted. From these results, we derive a new active minimal ribozyme that contains three helical elements, an internal loop, and a terminal loop. A three-dimensional model of this truncated ribozyme was generated using MC-SYM, and confirms that the catalytic core of the minimized construct can adopt a tertiary structure that is very similar to that of the nontruncated version. A new strategy is described to study the functional importance of various residues and chemical groups and to identify specific interdomain interactions. This approach uses two physically separated and truncated domains derived from the minimal motif.

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Section: Discussionsupporting

confidence: 89%

Modifications and deletions of helices within the hairpin ribozyme–substrate complex: An active ribozyme lacking helix 1

Pinard

Lambert²,

Pothiawala³

et al. 2004

RNA

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“…This ribozyme occurs naturally in the context of a four-way helical junction, although the minimum catalytically competent motif requires only two of these helices, each containing conserved loop domains that interact to form the active center (Ferre-D'Amare 2004). In the minimized version, the two helices are hinged at the junction point to allow association of the loop domains, and folding of this form requires significantly higher Mg 2+ concentrations as compared with the native form (Walter et al 1999;Zhao et al 2000). Thus, the interdomain junction of the hairpin ribozyme, which is remote from the catalytic core, nevertheless exerts a profound influence on the folding of this motif.…”

Section: Discussionmentioning

confidence: 99%

Characteristics of the glmS ribozyme suggest only structural roles for divalent metal ions

Roth

Nahvi²,

Lee³

et al. 2006

RNA

107

151

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The glmS ribozyme is a riboswitch class that occurs in certain Gram-positive bacteria, where it resides within mRNAs encoding glucosamine 6-phosphate synthase. Members of this self-cleaving ribozyme class rapidly catalyze RNA transesterification upon binding GlcN6P, and genetic evidence suggests that this cleavage event is important for down-regulating GlmS protein expression. In this report, we present a refined secondary structure model of the glmS ribozyme and determine the importance of a conserved pseudoknot structure for optimal ribozyme function. Analyses of deletion constructs demonstrate that the pseudoknot, together with other structural elements, permits the ribozyme to achieve maximum rate constants for RNA cleavage at physiologically relevant Mg 2+ concentrations. In addition, we show that substantial rate enhancements are supported by an exchange-inert cobalt (III) complex and by molar concentrations of monovalent ions. Our findings indicate that the glmS ribozyme forms a complex structure to employ catalytic strategies that do not require the direct participation of divalent metal ions.

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“…As has been pointed out (Khvorova et al 2003;Penedo et al 2004), the discovery of stabilizing elements in the HHRz parallels another small ribozyme. In the hairpin ribozyme, changing a two-or three-way junction to the native four-way junction increases catalytic activity in lower concentrations of Mg 2+ (Esteban et al 1997;Walter et al 1999;Zhao et al 2000), shifts the internal equilibrium toward ligation (Fedor 1999), and increases the rate of folding (Tan et al 2003).…”

Section: Discussionmentioning

confidence: 99%

Characterization of a native hammerhead ribozyme derived from schistosomes

Osborne

Schaak²,

DeRose³

2005

RNA

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A recent re-examination of the role of the helices surrounding the conserved core of the hammerhead ribozyme has identified putative loop-loop interactions between stems I and II in native hammerhead sequences. These extended hammerhead sequences are more active at low concentrations of divalent cations than are minimal hammerheads. The loop-loop interactions are proposed to stabilize a more active conformation of the conserved core. Here, a kinetic and thermodynamic characterization of an extended hammerhead sequence derived from Schistosoma mansoni is performed. Biphasic kinetics are observed, suggesting the presence of at least two conformers, one cleaving with a fast rate and the other with a slow rate. Replacing loop II with a poly(U) sequence designed to eliminate the interaction between the two loops results in greatly diminished activity, suggesting that the loop-loop interactions do aid in forming a more active conformation. Previous studies with minimal hammerheads have shown deleterious effects of R p -phosphorothioate substitutions at the cleavage site and 5 to A9, both of which could be rescued with Cd 2+ . Here, phosphorothioate modifications at the cleavage site and 5 to A9 were made in the schistosome-derived sequence. In Mg 2+ , both phosphorothioate substitutions decreased the overall fraction cleaved without significantly affecting the observed rate of cleavage. The addition of Cd 2+ rescued cleavage in both cases, suggesting that these are still putative metal binding sites in this native sequence.

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The folding of the hairpin ribozyme: Dependence on the loops and the junction

Cited by 57 publications

References 69 publications

Modifications and deletions of helices within the hairpin ribozyme–substrate complex: An active ribozyme lacking helix 1

Modifications and deletions of helices within the hairpin ribozyme–substrate complex: An active ribozyme lacking helix 1

Characteristics of the glmS ribozyme suggest only structural roles for divalent metal ions

Characterization of a native hammerhead ribozyme derived from schistosomes

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