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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 3. Schematic of the difference CD assay for the binding of ions linked to domain docking. In the absence of any docking, the observed CD spectrum of a mixture of loops A and B [Δε(A+B), solid line] will be the arithmetic sum of those for the individual loops (ΔεA and ΔεB, dashed lines) (top), whereas if docking occurs, a change in the ellipticity from that simple sum will be observed (shaded area, ΔΔε) (bottom). Circles indicate multivalent cations bound to RNA, shaded in the case of ions whose binding is linked to domain docking.
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Figure 3: Figure 3. Schematic of the difference CD assay for the binding of ions linked to domain docking. In the absence of any docking, the observed CD spectrum of a mixture of loops A and B [Δε(A+B), solid line] will be the arithmetic sum of those for the individual loops (ΔεA and ΔεB, dashed lines) (top), whereas if docking occurs, a change in the ellipticity from that simple sum will be observed (shaded area, ΔΔε) (bottom). Circles indicate multivalent cations bound to RNA, shaded in the case of ions whose binding is linked to domain docking.

Mentions: CD spectra of nucleic acids are sensitive to overall structural features, including base stacking, and have found use in studies of RNA and DNA structure and folding.56-58 The CD spectra of loops A and B of the hairpin ribozyme would be expected to be altered upon metal ion-driven domain docking, potentially providing a useful signature for the metal dependence of docking. In a simple titration of a mixture of the two loops with Co(NH3)63+, however, this effect would be obscured by signals arising from the extensive non-specific interactions of trivalent metal ions with the polyanionic RNA backbone. Indeed, CD spectra of isolated hairpin loops showed features consistent with A-form structure that increased in intensity upon equilibration with sub-millimolar amounts of Co(NH3)63+ (Fig. S1). We therefore devised a novel difference CD assay to isolate the cation-binding isotherm of only those metal ion(s) whose interaction with the RNA is linked to domain docking. In this procedure (Fig. 3), separate CD spectra at identical Co(NH3)63+ concentrations are acquired for three samples: Loop A alone, loop B alone and a 1:1 mixture of the two molecules. Samples must be carefully dialyzed against Co-containing buffer to ensure identical concentrations of free cation (see Materials and Methods). If no interaction between the two loops occurs, the CD signal for the 1:1 mixture [Δε(A+B)] will be identical to the numerical sum of the signals for the two isolated loops (ΔεA + ΔεB) (Fig. 3, upper panel). To the extent that the loops interact, however, the CD spectrum of the mixture will differ from the sum of the individual loops (Fig. 3, lower panel). The magnitude of the difference ΔΔε, taken at the wavelength peak of 270 nm, thus provides a direct readout of intermolecular domain docking. A plot of this parameter vs. [Co(NH3)63+] reveals an apparent cation-binding isotherm for only those cations whose binding is thermodynamically linked to domain docking. The difference spectrum, unlike the CD signals of the individual samples, is unaffected by RNA-cation interactions that occur independently of domain 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 3. Schematic of the difference CD assay for the binding of ions linked to domain docking. In the absence of any docking, the observed CD spectrum of a mixture of loops A and B [Δε(A+B), solid line] will be the arithmetic sum of those for the individual loops (ΔεA and ΔεB, dashed lines) (top), whereas if docking occurs, a change in the ellipticity from that simple sum will be observed (shaded area, ΔΔε) (bottom). Circles indicate multivalent cations bound to RNA, shaded in the case of ions whose binding is linked to domain docking.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Figure 3. Schematic of the difference CD assay for the binding of ions linked to domain docking. In the absence of any docking, the observed CD spectrum of a mixture of loops A and B [Δε(A+B), solid line] will be the arithmetic sum of those for the individual loops (ΔεA and ΔεB, dashed lines) (top), whereas if docking occurs, a change in the ellipticity from that simple sum will be observed (shaded area, ΔΔε) (bottom). Circles indicate multivalent cations bound to RNA, shaded in the case of ions whose binding is linked to domain docking.
Mentions: CD spectra of nucleic acids are sensitive to overall structural features, including base stacking, and have found use in studies of RNA and DNA structure and folding.56-58 The CD spectra of loops A and B of the hairpin ribozyme would be expected to be altered upon metal ion-driven domain docking, potentially providing a useful signature for the metal dependence of docking. In a simple titration of a mixture of the two loops with Co(NH3)63+, however, this effect would be obscured by signals arising from the extensive non-specific interactions of trivalent metal ions with the polyanionic RNA backbone. Indeed, CD spectra of isolated hairpin loops showed features consistent with A-form structure that increased in intensity upon equilibration with sub-millimolar amounts of Co(NH3)63+ (Fig. S1). We therefore devised a novel difference CD assay to isolate the cation-binding isotherm of only those metal ion(s) whose interaction with the RNA is linked to domain docking. In this procedure (Fig. 3), separate CD spectra at identical Co(NH3)63+ concentrations are acquired for three samples: Loop A alone, loop B alone and a 1:1 mixture of the two molecules. Samples must be carefully dialyzed against Co-containing buffer to ensure identical concentrations of free cation (see Materials and Methods). If no interaction between the two loops occurs, the CD signal for the 1:1 mixture [Δε(A+B)] will be identical to the numerical sum of the signals for the two isolated loops (ΔεA + ΔεB) (Fig. 3, upper panel). To the extent that the loops interact, however, the CD spectrum of the mixture will differ from the sum of the individual loops (Fig. 3, lower panel). The magnitude of the difference ΔΔε, taken at the wavelength peak of 270 nm, thus provides a direct readout of intermolecular domain docking. A plot of this parameter vs. [Co(NH3)63+] reveals an apparent cation-binding isotherm for only those cations whose binding is thermodynamically linked to domain docking. The difference spectrum, unlike the CD signals of the individual samples, is unaffected by RNA-cation interactions that occur independently of domain 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