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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 7. Cleavage kinetics of the trans-docking hairpin ribozyme at 250 μM Co(NH3)63+. Top, 20% polyacrylamide gel analysis of the cleavage of 3′-32P-labeled loop A RNA, demonstrating the lack of starting material present at long time points. S, uncleaved loop A substrate (26 nucleotides); P, 3′ cleavage product (21 nucleotides). Bottom, densitometric scan of the gel data fit to a single rising exponential. For this data set, the extrapolated cleavage at infinite time is 86.1%.
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Figure 7: Figure 7. Cleavage kinetics of the trans-docking hairpin ribozyme at 250 μM Co(NH3)63+. Top, 20% polyacrylamide gel analysis of the cleavage of 3′-32P-labeled loop A RNA, demonstrating the lack of starting material present at long time points. S, uncleaved loop A substrate (26 nucleotides); P, 3′ cleavage product (21 nucleotides). Bottom, densitometric scan of the gel data fit to a single rising exponential. For this data set, the extrapolated cleavage at infinite time is 86.1%.

Mentions: The metal activation of hairpin ribozyme self-cleavage in two- and four-way junction constructs has been extensively studied (see Introduction). In situations including the activation of a four-way junction construct with various metal ions, distinct ion dependencies for folding and cleavage have been reported.20,59 The use of an intermolecular docking system in the present work allows a straightforward comparison of the metal dependence of domain docking, as determined above using CD, with that of ribozyme self-cleavage. These measurements take place in the absence of effects mediated by metal interactions with the junction. By considering the loop B construct as the enzyme and the loop A construct as the substrate, we determined the rate of ribozyme reaction kobs under single-turnover conditions (excess of loop B over loop A) as a function of Co(NH3)63+ concentration. In all cases, essentially complete cleavage of loop A was observed at long time points (Fig. 7), allowing us to consider the undocking of cleaved loop A from loop B and/or the dissociation of cleavage products to be essentially irreversible. We thus neglected the effects of back-reaction in the kinetic analysis (see below). We performed most of our measurements at 500 nM loop B, which is only slightly above the Kd for the two loops determined above. However, when the 350 μM Co(NH3)63+ reaction was repeated at a loop B concentration of 3 μM, the resulting rate was identical within error to that observed at 500 nM loop B (data not shown), indicating that saturating concentrations of loop B had been reached. Consistent with this, a 10-fold lower apparent Km vs. docking Kd was observed by Burke and co-workers,55 possibly attributable to the difference in chemical structure at the nucleophilic oxygen (see above). At saturating concentrations of cation, kobs reached approximately 0.1 min−1 (extrapolated value 0.113 ± 0.027 min−1), consistent with limiting values reached in previous studies of this format.25,55 No cleavage was observed for the control G+1A mutant of loop A (data not shown).


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 7. Cleavage kinetics of the trans-docking hairpin ribozyme at 250 μM Co(NH3)63+. Top, 20% polyacrylamide gel analysis of the cleavage of 3′-32P-labeled loop A RNA, demonstrating the lack of starting material present at long time points. S, uncleaved loop A substrate (26 nucleotides); P, 3′ cleavage product (21 nucleotides). Bottom, densitometric scan of the gel data fit to a single rising exponential. For this data set, the extrapolated cleavage at infinite time is 86.1%.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672286&req=5

Figure 7: Figure 7. Cleavage kinetics of the trans-docking hairpin ribozyme at 250 μM Co(NH3)63+. Top, 20% polyacrylamide gel analysis of the cleavage of 3′-32P-labeled loop A RNA, demonstrating the lack of starting material present at long time points. S, uncleaved loop A substrate (26 nucleotides); P, 3′ cleavage product (21 nucleotides). Bottom, densitometric scan of the gel data fit to a single rising exponential. For this data set, the extrapolated cleavage at infinite time is 86.1%.
Mentions: The metal activation of hairpin ribozyme self-cleavage in two- and four-way junction constructs has been extensively studied (see Introduction). In situations including the activation of a four-way junction construct with various metal ions, distinct ion dependencies for folding and cleavage have been reported.20,59 The use of an intermolecular docking system in the present work allows a straightforward comparison of the metal dependence of domain docking, as determined above using CD, with that of ribozyme self-cleavage. These measurements take place in the absence of effects mediated by metal interactions with the junction. By considering the loop B construct as the enzyme and the loop A construct as the substrate, we determined the rate of ribozyme reaction kobs under single-turnover conditions (excess of loop B over loop A) as a function of Co(NH3)63+ concentration. In all cases, essentially complete cleavage of loop A was observed at long time points (Fig. 7), allowing us to consider the undocking of cleaved loop A from loop B and/or the dissociation of cleavage products to be essentially irreversible. We thus neglected the effects of back-reaction in the kinetic analysis (see below). We performed most of our measurements at 500 nM loop B, which is only slightly above the Kd for the two loops determined above. However, when the 350 μM Co(NH3)63+ reaction was repeated at a loop B concentration of 3 μM, the resulting rate was identical within error to that observed at 500 nM loop B (data not shown), indicating that saturating concentrations of loop B had been reached. Consistent with this, a 10-fold lower apparent Km vs. docking Kd was observed by Burke and co-workers,55 possibly attributable to the difference in chemical structure at the nucleophilic oxygen (see above). At saturating concentrations of cation, kobs reached approximately 0.1 min−1 (extrapolated value 0.113 ± 0.027 min−1), consistent with limiting values reached in previous studies of this format.25,55 No cleavage was observed for the control G+1A mutant of loop A (data not shown).

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