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