5 research outputs found
Monovalent ions modulate the flux through multiple folding pathways of an RNA pseudoknot
The functions of RNA pseudoknots (PKs), which are minimal tertiary structural
motifs and an integral part of several ribozymes and ribonucleoprotein
complexes, are determined by their structure, stability and dynamics.
Therefore, it is important to elucidate the general principles governing their
thermodynamics/folding mechanisms. Here, we combine experiments and simulations
to examine the folding/unfolding pathways of the VPK pseudoknot, a variant of
the Mouse Mammary Tumor Virus (MMTV) PK involved in ribosomal frameshifting.
Fluorescent nucleotide analogs (2-aminopurine and pyrrolocytidine) placed at
different stem/loop positions in the PK, and laser temperature-jump approaches
serve as local probes allowing us to monitor the order of assembly of VPK with
two helices with different intrinsic stabilities. The experiments and molecular
simulations show that at 50 mM KCl the dominant folding pathway populates only
the more stable partially folded hairpin. As the salt concentration is
increased a parallel folding pathway emerges, involving the less stable hairpin
structure as an alternate intermediate. Notably, the flux between the pathways
is modulated by the ionic strength. The findings support the principle that the
order of PK structure formation is determined by the relative stabilities of
the hairpins, which can be altered by sequence variations or salt
concentrations. Our study not only unambiguously demonstrates that PK folds by
parallel pathways, but also establishes that quantitative description of RNA
self-assembly requires a synergistic combination of experiments and
simulations.Comment: Supporting Information include
Exploring the energy landscape of nucleic acid hairpins using laser temperature-jump and microfluidic mixing
We have investigated the multidimensionality of the free energy landscape accessible to a nucleic acid hairpin by measuring the relaxation kinetics in response to two very different perturbations of the folding/unfolding equilibrium, either a laser temperature-jump or ion-jump (from rapid mixing with counterions). The two sets of measurements carried out on DNA hairpins (4 or 5 base pairs in the stem and 21-nucleotide polythymine loop), using FRET between end labels or fluorescence of 2-aminopurine in the stem as conformational probes, yield distinctly different relaxation kinetics in the temperature range 10–30 °C and salt range 100–500 mM NaCl, with rapid mixing exhibiting slower relaxation kinetics after an initial collapse of the chain within 8 μs of the counterion mixing time. The discrepancy in the relaxation times increases with increasing temperatures, with rapid mixing times nearly 10-fold slower than T-jump times at 30 °C. These results rule out a simple two-state scenario with the folded and unfolded ensemble separated by a significant free energy barrier, even at temperatures close to the thermal melting temperature Tm. Instead, our results point to the scenario in which the conformational ensemble accessed after counterion condensation and collapse of the chain is distinctly different from the unfolded ensemble accessed with T-jump perturbation. Our data suggest that, even at temperatures in the vicinity of Tm or higher, the relaxation kinetics obtained from the ion-jump measurements are dominated by the escape from the collapsed state accessed after counterion condensation
Exploring the Energy Landscape of Nucleic Acid Hairpins Using Laser Temperature-Jump and Microfluidic Mixing
We have investigated the multidimensionality of the free
energy
landscape accessible to a nucleic acid hairpin by measuring the relaxation
kinetics in response to two very different perturbations of the folding/unfolding
equilibrium, either a laser temperature-jump or ion-jump (from rapid
mixing with counterions). The two sets of measurements carried out
on DNA hairpins (4 or 5 base pairs in the stem and 21-nucleotide polythymine
loop), using FRET between end labels or fluorescence of 2-aminopurine
in the stem as conformational probes, yield distinctly different relaxation
kinetics in the temperature range 10–30 °C and salt range
100–500 mM NaCl, with rapid mixing exhibiting slower relaxation
kinetics after an initial collapse of the chain within 8 μs
of the counterion mixing time. The discrepancy in the relaxation times
increases with increasing temperatures, with rapid mixing times nearly
10-fold slower than T-jump times at 30 °C. These results rule
out a simple two-state scenario with the folded and unfolded ensemble
separated by a significant free energy barrier, even at temperatures
close to the thermal melting temperature <i>T</i><sub>m</sub>. Instead, our results point to the scenario in which the conformational
ensemble accessed after counterion condensation and collapse of the
chain is distinctly different from the unfolded ensemble accessed
with T-jump perturbation. Our data suggest that, even at temperatures
in the vicinity of <i>T</i><sub>m</sub> or higher, the relaxation
kinetics obtained from the ion-jump measurements are dominated by
the escape from the collapsed state accessed after counterion condensation