1998
DOI: 10.1021/bi981513+
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Structure and Activity of the Hairpin Ribozyme in Its Natural Junction Conformation: Effect of Metal Ions

Abstract: 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… Show more

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Cited by 79 publications
(62 citation statements)
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“…The overall topology structure of the extended kissing complex is similar to some other RNA four-way junctions (Krol et al+, 1990;Walter et al+, 1998a;Nowakowski et al+, 1999) and DNA Holliday junctions (Duckett et al+, 1992)+ In both DNA and RNA four-way junctions, divalent ions are required for the formation and stabilization of antiparallel X-shaped structures (Duckett et al+, 1992;Walter et al+, 1998b)+ The particularity of the proposed CopA-CopT structure is the crossing over of the strands at the junction under the constraints imposed by the two loops connecting intermolecular helices B and B9+ This forces a side-by-side alignment of the two helical domains that brings the 59 tail of CopA in close proximity to the complementary region of CopT (Fig+ 7)+ The formation of intermolecular helix C, which clamps the two long helical domains, greatly enhances the stability of the complex (Persson et al+, 1990a;Malmgren et al+, 1997)+ Crystallographic analysis of a group I ribozyme domain revealed a similar organization (Cate et al+, 1996): a sharp bend induced by an internal loop allows a side-by-side alignment of two helical domains that is additionally stabilized by metal-and ribosemediated backbone contacts and two long-range tertiary interactions+ A side-by-side configuration was also proposed for the hairpin ribozyme, here stabilized by interactions between two internal loops (Earnshaw et al+, 1997)+ The formation of a stable RNA-RNA complex is not unique to CopA-CopT, and is also a key feature in the replication control of plasmids belonging to the IncB and IncIa groups (Siemering et al+, 1994; plasmids+ In these systems, the antisense RNAs inhibit the formation of a pseudoknot structure that activates rep translation (Wilson et al+, 1993;)+ All these antisense and target RNAs are characterized by stable hairpins with identical loop sequences and bulged residues in the upper stem regions+ Enzymatic probing performed on (antisense) RNAI in pMU720 plasmid bound to its target indicated that a full duplex was not rapidly formed in vitro+ Instead, binding resulted in an extended kissing complex stabilized by 59 tail interactions (Siemering et al+, 1994)+ One may therefore speculate that, in all these systems, the final product of the binding reaction in vitro is characterized by an overall topology very similar to that reported here, except that the lengths of helices B and B9, if formed in the IncB/IncIa cases, could be different+…”
Section: Conversion Of a Loop-loop Interaction To A Four-helix Junctionmentioning
confidence: 52%
“…The overall topology structure of the extended kissing complex is similar to some other RNA four-way junctions (Krol et al+, 1990;Walter et al+, 1998a;Nowakowski et al+, 1999) and DNA Holliday junctions (Duckett et al+, 1992)+ In both DNA and RNA four-way junctions, divalent ions are required for the formation and stabilization of antiparallel X-shaped structures (Duckett et al+, 1992;Walter et al+, 1998b)+ The particularity of the proposed CopA-CopT structure is the crossing over of the strands at the junction under the constraints imposed by the two loops connecting intermolecular helices B and B9+ This forces a side-by-side alignment of the two helical domains that brings the 59 tail of CopA in close proximity to the complementary region of CopT (Fig+ 7)+ The formation of intermolecular helix C, which clamps the two long helical domains, greatly enhances the stability of the complex (Persson et al+, 1990a;Malmgren et al+, 1997)+ Crystallographic analysis of a group I ribozyme domain revealed a similar organization (Cate et al+, 1996): a sharp bend induced by an internal loop allows a side-by-side alignment of two helical domains that is additionally stabilized by metal-and ribosemediated backbone contacts and two long-range tertiary interactions+ A side-by-side configuration was also proposed for the hairpin ribozyme, here stabilized by interactions between two internal loops (Earnshaw et al+, 1997)+ The formation of a stable RNA-RNA complex is not unique to CopA-CopT, and is also a key feature in the replication control of plasmids belonging to the IncB and IncIa groups (Siemering et al+, 1994; plasmids+ In these systems, the antisense RNAs inhibit the formation of a pseudoknot structure that activates rep translation (Wilson et al+, 1993;)+ All these antisense and target RNAs are characterized by stable hairpins with identical loop sequences and bulged residues in the upper stem regions+ Enzymatic probing performed on (antisense) RNAI in pMU720 plasmid bound to its target indicated that a full duplex was not rapidly formed in vitro+ Instead, binding resulted in an extended kissing complex stabilized by 59 tail interactions (Siemering et al+, 1994)+ One may therefore speculate that, in all these systems, the final product of the binding reaction in vitro is characterized by an overall topology very similar to that reported here, except that the lengths of helices B and B9, if formed in the IncB/IncIa cases, could be different+…”
Section: Conversion Of a Loop-loop Interaction To A Four-helix Junctionmentioning
confidence: 52%
“…For example, inspection of the structures of the 16S and 23S rRNA species within the ribosome reveals many helical junctions (Ban et al 2000;Wimberly et al 2000). The structure of some autonomously folding smaller RNA species can be almost entirely determined by component junctions Walter et al 1998), as exemplified by the VS ribozyme where the structural core of the ribozyme comprises five helical segments related by two three-way junctions (Lafontaine et al 2002;Lipfert et al 2008). Helical junctions can also generate the catalytic center of ribozymes, such as the hammerhead ribozyme (Martick et al 2008), and ligand binding pockets of great selectivity, as found in a number of riboswitches (Serganov et al 2004(Serganov et al , 2008Garst et al 2008).…”
Section: Introductionmentioning
confidence: 99%
“…The helical arms of junctions in both DNA and RNA have a strong propensity to undergo coaxial stacking when electrostatic repulsion is minimized by the presence of metal ions (Duckett et al 1988(Duckett et al , 1995Orr et al 1998;Walter et al 1998; Lescoute and Westhof 2006;de la Pena et al 2009). In the case of three-way junctions, only two arms can be stacked in this way, and the third must remain unstacked.…”
Section: Introductionmentioning
confidence: 99%
“…Hairpin ribozymes are usually more efficient at 37°C than hammerhead ribozymes and cofactors are not a strict requirement for activity because the catalytic mechanism appears to rely on structural components (Walter et al, 1998 …”
Section: Hairpin Ribozymes and Hpvmentioning
confidence: 99%