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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 6. (A) Unreferenced surface plasmon resonance data acquired at 250 μM Co(NH3)63+ and 450 nM loop B. Upper trace, wild-type loop A; lower trace, G+1A mutant loop A immobilized on the same chip. (B) Representative fully referenced SPR analysis of domain docking at 250 μM Co(NH3)63+. Data shown are differences between a channel with immobilized wild-type loop A and one with immobilized G+1A mutant (compare left panel) and, thus, reflect only specific docking interactions. Red lines are global fits to a 1:1 interaction model incorporating bulk refractive-index shift. Concentrations of loop B used: 150 nM, 300 nM, 450 nM, 600 nM, 750 nM.
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Figure 6: Figure 6. (A) Unreferenced surface plasmon resonance data acquired at 250 μM Co(NH3)63+ and 450 nM loop B. Upper trace, wild-type loop A; lower trace, G+1A mutant loop A immobilized on the same chip. (B) Representative fully referenced SPR analysis of domain docking at 250 μM Co(NH3)63+. Data shown are differences between a channel with immobilized wild-type loop A and one with immobilized G+1A mutant (compare left panel) and, thus, reflect only specific docking interactions. Red lines are global fits to a 1:1 interaction model incorporating bulk refractive-index shift. Concentrations of loop B used: 150 nM, 300 nM, 450 nM, 600 nM, 750 nM.

Mentions: For analysis of the kinetics of hairpin ribozyme loop-loop interactions in the trans-docking format, we used a surface plasmon resonance (SPR) assay to monitor the formation of the bound complex in real time. Chemically synthesized 5′-biotinylated wild-type loop A, rendered unreactive with a 2’-O-methyl modification at the nucleophilic group, was captured on one of the two SPR flow cells. Loop A with a G+1A mutation that renders the molecule inactive for both docking and catalysis was captured on the second channel as a reference. Figure 6A shows representative raw wild-type and mutant injection data. Both species were affected by a rapidly appearing bulk shift in refractive index, which we speculate arose from increased local concentration of high-mass cobalt complexes. A much slower, authentic macromolecular binding event was clearly present in channels containing wild-type loop A but absent in the case of the non-docking G+1A mutant. Given the dramatic difference in results upon this highly conservative sequence change, we interpret the slower event as representing the specific formation of hairpin ribozyme tertiary structure via intermolecular domain binding.


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 6. (A) Unreferenced surface plasmon resonance data acquired at 250 μM Co(NH3)63+ and 450 nM loop B. Upper trace, wild-type loop A; lower trace, G+1A mutant loop A immobilized on the same chip. (B) Representative fully referenced SPR analysis of domain docking at 250 μM Co(NH3)63+. Data shown are differences between a channel with immobilized wild-type loop A and one with immobilized G+1A mutant (compare left panel) and, thus, reflect only specific docking interactions. Red lines are global fits to a 1:1 interaction model incorporating bulk refractive-index shift. Concentrations of loop B used: 150 nM, 300 nM, 450 nM, 600 nM, 750 nM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Figure 6. (A) Unreferenced surface plasmon resonance data acquired at 250 μM Co(NH3)63+ and 450 nM loop B. Upper trace, wild-type loop A; lower trace, G+1A mutant loop A immobilized on the same chip. (B) Representative fully referenced SPR analysis of domain docking at 250 μM Co(NH3)63+. Data shown are differences between a channel with immobilized wild-type loop A and one with immobilized G+1A mutant (compare left panel) and, thus, reflect only specific docking interactions. Red lines are global fits to a 1:1 interaction model incorporating bulk refractive-index shift. Concentrations of loop B used: 150 nM, 300 nM, 450 nM, 600 nM, 750 nM.
Mentions: For analysis of the kinetics of hairpin ribozyme loop-loop interactions in the trans-docking format, we used a surface plasmon resonance (SPR) assay to monitor the formation of the bound complex in real time. Chemically synthesized 5′-biotinylated wild-type loop A, rendered unreactive with a 2’-O-methyl modification at the nucleophilic group, was captured on one of the two SPR flow cells. Loop A with a G+1A mutation that renders the molecule inactive for both docking and catalysis was captured on the second channel as a reference. Figure 6A shows representative raw wild-type and mutant injection data. Both species were affected by a rapidly appearing bulk shift in refractive index, which we speculate arose from increased local concentration of high-mass cobalt complexes. A much slower, authentic macromolecular binding event was clearly present in channels containing wild-type loop A but absent in the case of the non-docking G+1A mutant. Given the dramatic difference in results upon this highly conservative sequence change, we interpret the slower event as representing the specific formation of hairpin ribozyme tertiary structure via intermolecular domain binding.

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