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Intermolecular domain docking in the hairpin ribozyme: metal dependence, binding kinetics and catalysis.

Sumita M, White NA, Julien KR, Hoogstraten CG - RNA Biol (2013)

Bottom Line: These two loops interact in a cation-driven docking step prior to chemical catalysis to form a tightly integrated structure, with dramatic changes occurring in the conformation of each loop upon docking.RNA self-cleavage requires binding of lower-affinity ions with greater apparent cooperativity than the docking process itself, implying that, even in the absence of direct coordination to RNA, metal ions play a catalytic role in hairpin ribozyme function beyond simply driving loop-loop docking.This observation is consistent with a "double conformational capture" model in which only collisions between loop A and loop B molecules that are simultaneously in minor, docking-competent conformations are productive for binding.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology; Michigan State University; East Lansing, MI USA.

ABSTRACT
The hairpin ribozyme is a prototype small, self-cleaving RNA motif. It exists naturally as a four-way RNA junction containing two internal loops on adjoining arms. These two loops interact in a cation-driven docking step prior to chemical catalysis to form a tightly integrated structure, with dramatic changes occurring in the conformation of each loop upon docking. We investigate the thermodynamics and kinetics of the docking process using constructs in which loop A and loop B reside on separate molecules. Using a novel CD difference assay to isolate the effects of metal ions linked to domain docking, we find the intermolecular docking process to be driven by sub-millimolar concentrations of the exchange-inert Co(NH 3) 6 (3+). RNA self-cleavage requires binding of lower-affinity ions with greater apparent cooperativity than the docking process itself, implying that, even in the absence of direct coordination to RNA, metal ions play a catalytic role in hairpin ribozyme function beyond simply driving loop-loop docking. Surface plasmon resonance assays reveal remarkably slow molecular association, given the relatively tight loop-loop interaction. This observation is consistent with a "double conformational capture" model in which only collisions between loop A and loop B molecules that are simultaneously in minor, docking-competent conformations are productive for binding.

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Related in: MedlinePlus

Figure 1. Hairpin ribozyme loop A and loop B constructs used in the present work shown with their context in the native, four-way junction ribozyme. Arrow indicates the site of self-cleavage.
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Figure 1: Figure 1. Hairpin ribozyme loop A and loop B constructs used in the present work shown with their context in the native, four-way junction ribozyme. Arrow indicates the site of self-cleavage.

Mentions: The hairpin ribozyme exists naturally with loops A and B on adjoining arms of an RNA four-way junction, the presence of which greatly favors the formation of the docked state (Fig. 1).19-21 The ribozyme is also highly active in a two-way junction or “minimal” form, on which much of the biochemical work has been performed.22,23 Finally, cleavage activity has been observed with internal loops A and B on separate molecules24,25 (Fig. 1). In this junctionless form, which we refer to as the “trans-docking” version of the ribozyme, the docking transition represents the formation of an RNA tertiary-structure interaction in isolation from hybridization or other effects.


Intermolecular domain docking in the hairpin ribozyme: metal dependence, binding kinetics and catalysis.

Sumita M, White NA, Julien KR, Hoogstraten CG - RNA Biol (2013)

Figure 1. Hairpin ribozyme loop A and loop B constructs used in the present work shown with their context in the native, four-way junction ribozyme. Arrow indicates the site of self-cleavage.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672286&req=5

Figure 1: Figure 1. Hairpin ribozyme loop A and loop B constructs used in the present work shown with their context in the native, four-way junction ribozyme. Arrow indicates the site of self-cleavage.
Mentions: The hairpin ribozyme exists naturally with loops A and B on adjoining arms of an RNA four-way junction, the presence of which greatly favors the formation of the docked state (Fig. 1).19-21 The ribozyme is also highly active in a two-way junction or “minimal” form, on which much of the biochemical work has been performed.22,23 Finally, cleavage activity has been observed with internal loops A and B on separate molecules24,25 (Fig. 1). In this junctionless form, which we refer to as the “trans-docking” version of the ribozyme, the docking transition represents the formation of an RNA tertiary-structure interaction in isolation from hybridization or other effects.

Bottom Line: These two loops interact in a cation-driven docking step prior to chemical catalysis to form a tightly integrated structure, with dramatic changes occurring in the conformation of each loop upon docking.RNA self-cleavage requires binding of lower-affinity ions with greater apparent cooperativity than the docking process itself, implying that, even in the absence of direct coordination to RNA, metal ions play a catalytic role in hairpin ribozyme function beyond simply driving loop-loop docking.This observation is consistent with a "double conformational capture" model in which only collisions between loop A and loop B molecules that are simultaneously in minor, docking-competent conformations are productive for binding.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology; Michigan State University; East Lansing, MI USA.

ABSTRACT
The hairpin ribozyme is a prototype small, self-cleaving RNA motif. It exists naturally as a four-way RNA junction containing two internal loops on adjoining arms. These two loops interact in a cation-driven docking step prior to chemical catalysis to form a tightly integrated structure, with dramatic changes occurring in the conformation of each loop upon docking. We investigate the thermodynamics and kinetics of the docking process using constructs in which loop A and loop B reside on separate molecules. Using a novel CD difference assay to isolate the effects of metal ions linked to domain docking, we find the intermolecular docking process to be driven by sub-millimolar concentrations of the exchange-inert Co(NH 3) 6 (3+). RNA self-cleavage requires binding of lower-affinity ions with greater apparent cooperativity than the docking process itself, implying that, even in the absence of direct coordination to RNA, metal ions play a catalytic role in hairpin ribozyme function beyond simply driving loop-loop docking. Surface plasmon resonance assays reveal remarkably slow molecular association, given the relatively tight loop-loop interaction. This observation is consistent with a "double conformational capture" model in which only collisions between loop A and loop B molecules that are simultaneously in minor, docking-competent conformations are productive for binding.

Show MeSH
Related in: MedlinePlus