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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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Figure 8. (A) Metal-dependent kinetics of the hairpin cleavage reaction in trans-docking format. Solid line is the fit to Equation 2, whereas the dashed line is the best-fit CD analysis of docking from Figure 5 scaled to the same limiting y-value at (Co)→∞ as the kinetic data. (B) Kinetic data shown as a Hill plot. (C) Kinetic scheme for the reaction in trans-docking form indicating the observed irreversibility of undocking and/or dissociation of cleavage products (see text). Rate constants ka and kd correspond to those measured using SPR in this work.
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Figure 8: Figure 8. (A) Metal-dependent kinetics of the hairpin cleavage reaction in trans-docking format. Solid line is the fit to Equation 2, whereas the dashed line is the best-fit CD analysis of docking from Figure 5 scaled to the same limiting y-value at (Co)→∞ as the kinetic data. (B) Kinetic data shown as a Hill plot. (C) Kinetic scheme for the reaction in trans-docking form indicating the observed irreversibility of undocking and/or dissociation of cleavage products (see text). Rate constants ka and kd correspond to those measured using SPR in this work.

Mentions: Our measurements showed a strongly sigmoidal dependence of kobs on [Co(NH3)63+], implying cooperative binding of multiple ions necessary for cleavage (Fig. 8). Fitting with Equation 2 gave a (Co)½chem of 168 ± 51 μM. This value is significantly higher than the (Co)½dock observed above for interdomain docking as assayed by CD, implying that metal ions play a role in hairpin catalysis in this format beyond simply driving the trans-docking interaction. Consistent with this interpretation, the corresponding Hill plot exhibited linear behavior throughout the measured range with slope n of 1.87 ± 0.15, contrasting with the non-cooperative behavior observed for docking.


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 8. (A) Metal-dependent kinetics of the hairpin cleavage reaction in trans-docking format. Solid line is the fit to Equation 2, whereas the dashed line is the best-fit CD analysis of docking from Figure 5 scaled to the same limiting y-value at (Co)→∞ as the kinetic data. (B) Kinetic data shown as a Hill plot. (C) Kinetic scheme for the reaction in trans-docking form indicating the observed irreversibility of undocking and/or dissociation of cleavage products (see text). Rate constants ka and kd correspond to those measured using SPR in this work.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Figure 8. (A) Metal-dependent kinetics of the hairpin cleavage reaction in trans-docking format. Solid line is the fit to Equation 2, whereas the dashed line is the best-fit CD analysis of docking from Figure 5 scaled to the same limiting y-value at (Co)→∞ as the kinetic data. (B) Kinetic data shown as a Hill plot. (C) Kinetic scheme for the reaction in trans-docking form indicating the observed irreversibility of undocking and/or dissociation of cleavage products (see text). Rate constants ka and kd correspond to those measured using SPR in this work.
Mentions: Our measurements showed a strongly sigmoidal dependence of kobs on [Co(NH3)63+], implying cooperative binding of multiple ions necessary for cleavage (Fig. 8). Fitting with Equation 2 gave a (Co)½chem of 168 ± 51 μM. This value is significantly higher than the (Co)½dock observed above for interdomain docking as assayed by CD, implying that metal ions play a role in hairpin catalysis in this format beyond simply driving the trans-docking interaction. Consistent with this interpretation, the corresponding Hill plot exhibited linear behavior throughout the measured range with slope n of 1.87 ± 0.15, contrasting with the non-cooperative behavior observed for docking.

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