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Monovalent ions modulate the flux through multiple folding pathways of an RNA pseudoknot.

ABSTRACT: 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 laser temperature-jump experiments and coarse-grained simulations to determine the folding/unfolding pathways of VPK, 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 serve as local probes allowing us to monitor the order of assembly of VPK that has two constituent hairpins with different intrinsic stabilities. We show that at 50 mM KCl, the dominant folding pathway populates only the more stable hairpin intermediate; as the salt concentration is increased, a parallel folding pathway emerges involving the less stable hairpin as an alternate intermediate. Notably, the flux between the pathways is modulated by the ionic strength. Our 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. The experimental results of salt effects on the partitioning between the two folding pathways are in remarkable agreement with simulations that were performed with no adjustable parameters. Our study not only unambiguously demonstrates that VPK folds by parallel pathways but also showcases the power of combining experiments and simulations for a more enriched description of RNA self-assembly.

SUBMITTER: Roca J

PROVIDER: S-EPMC6077692 | biostudies-other | 2018 Jul

REPOSITORIES: biostudies-other

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Monovalent ions modulate the flux through multiple folding pathways of an RNA pseudoknot.

Roca Jorjethe J Hori Naoto N Baral Saroj S Velmurugu Yogambigai Y Narayanan Ranjani R Narayanan Prasanth P Thirumalai D D Ansari Anjum A

Proceedings of the National Academy of Sciences of the United States of America 20180716 31

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 laser temperature-jump experiments and coarse-grained simulations to determine the folding/unfolding pathways of VPK, a variant of the mouse mammary ...[more]

PMID: 30012621

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Project description:The effect of monovalent ions on both the reactivity and global folding of the 8-17 DNAzyme is investigated, and the results are compared with those of the hammerhead ribozyme, which has similar size and secondary structure. In contrast to the hammerhead ribozyme, the 8-17 DNAzyme activity is not detectable in the presence of 4 M K(+), Rb(+), or Cs(+) or in the presence of 80 mM, [Co(NH(3))(6)](3+). Only 4 M Li(+), NH(4)(+) and, to a lesser extent, Na(+) conferred detectable activity. The observed rate constants (k(obs) approximately 10(-3) min(-1) for Li(+) and NH(4)(+)) are approximately 1000-fold lower than that in the presence of 10 mM Mg(2+), and approximately 200,000-fold slower than that in the presence of 100 microM Pb(2+). Since the hammerhead ribozyme displays monovalent ion-dependent activity that is often within approximately 10-fold of divalent metal ion-dependent activity, these results suggest that the 8-17 DNAzyme, obtained by in vitro selections, has evolved to have a more stringent divalent metal ion requirement for high activity as compared to the naturally occurring ribozymes, making the 8-17 DNAzyme an excellent choice as a Pb(2+) sensor with high selectivity. In contrast to the activity data, folding was observed in the presence of all the monovalent ions investigated, although those monovalent ions that do not support DNAzyme activity have weaker binding affinity (K(d) approximately 0.35 M for Rb(+) and Cs(+)), while those that confer DNAzyme activity possess stronger affinity (K(d) approximately 0.22 M for Li(+), Na(+) and NH(4)(+)). In addition, a correlation between metal ion charge density, binding affinity and enzyme activity was found among mono- and divalent metal ions except Pb(2+); higher charge density resulted in stronger affinity and higher activity, suggesting that the observed folding and activity is at least partially due to electrostatic interactions between ions and the DNAzyme. Finally, circular dichroism (CD) study has revealed Z-DNA formation with the monovalent metal ions, Zn(2+) and Mg(2+); the K(d) values obtained using CD were in the same range as those obtained from folding studies using FRET. However, Z-DNA formation was not observed with Pb(2+). These results indicate that Pb(2+)-dependent function follows a different mechanism from the monovalent metal ions and other divalent metal ions; in the presence of latter metal ions, metal-ion dependent folding and structural changes, including formation of Z-DNA, play an important role in the catalytic function of the 8-17 DNAzyme.

Dataset Information

Monovalent ions modulate the flux through multiple folding pathways of an RNA pseudoknot.

Publications

Monovalent ions modulate the flux through multiple folding pathways of an RNA pseudoknot.

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