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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 4. Comparison of CD spectra for an equimolar mixture of loops A and B [Δε(A+B), solid line] with the arithmetic sum of spectra for the individual loops (ΔεA + ΔεB, dashed line). The difference between these traces (ΔΔε, dotted line) is taken as a measure of the extent of ion-driven domain docking. (A) Absence of Co(NH3)63+; (B) 250 μM-free Co(NH3)63+.
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Figure 4: Figure 4. Comparison of CD spectra for an equimolar mixture of loops A and B [Δε(A+B), solid line] with the arithmetic sum of spectra for the individual loops (ΔεA + ΔεB, dashed line). The difference between these traces (ΔΔε, dotted line) is taken as a measure of the extent of ion-driven domain docking. (A) Absence of Co(NH3)63+; (B) 250 μM-free Co(NH3)63+.

Mentions: Representative data obtained under two ionic conditions are shown in Figure 4. In the absence of Co(NH3)63+, domain docking was not expected to occur, and the spectrum of the mixed sample was indeed essentially identical to the sum of the spectra of the isolated loops throughout the region of the ellipticity maximum. Thus, ΔΔε measurements at a particular [Co(NH3)63+] may be presumed to arise from the effects of metal ions only. In the presence of 250 μM-free Co(NH3)63+, by contrast, a substantial shift between the two spectra was observed, leading to an easily quantifiable difference spectrum.


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 4. Comparison of CD spectra for an equimolar mixture of loops A and B [Δε(A+B), solid line] with the arithmetic sum of spectra for the individual loops (ΔεA + ΔεB, dashed line). The difference between these traces (ΔΔε, dotted line) is taken as a measure of the extent of ion-driven domain docking. (A) Absence of Co(NH3)63+; (B) 250 μM-free Co(NH3)63+.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Figure 4. Comparison of CD spectra for an equimolar mixture of loops A and B [Δε(A+B), solid line] with the arithmetic sum of spectra for the individual loops (ΔεA + ΔεB, dashed line). The difference between these traces (ΔΔε, dotted line) is taken as a measure of the extent of ion-driven domain docking. (A) Absence of Co(NH3)63+; (B) 250 μM-free Co(NH3)63+.
Mentions: Representative data obtained under two ionic conditions are shown in Figure 4. In the absence of Co(NH3)63+, domain docking was not expected to occur, and the spectrum of the mixed sample was indeed essentially identical to the sum of the spectra of the isolated loops throughout the region of the ellipticity maximum. Thus, ΔΔε measurements at a particular [Co(NH3)63+] may be presumed to arise from the effects of metal ions only. In the presence of 250 μM-free Co(NH3)63+, by contrast, a substantial shift between the two spectra was observed, leading to an easily quantifiable difference spectrum.

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