Free Energy Landscape and Multiple Folding Pathways of an H-Type RNA Pseudoknot

<div><p>How RNA sequences fold to specific tertiary structures is one of the key problems for understanding their dynamics and functions. Here, we study the folding process of an H-type RNA pseudoknot by performing a large-scale all-atom MD simulation and bias-exchange metadynamics. The folding free energy landscapes are obtained and several folding intermediates are identified. It is suggested that the folding occurs via multiple mechanisms, including a step-wise mechanism starting either from the first helix or the second, and a cooperative mechanism with both helices forming simultaneously. Despite of the multiple mechanism nature, the ensemble folding kinetics estimated from a Markov state model is single-exponential. It is also found that the correlation between folding and binding of metal ions is significant, and the bound ions mediate long-range interactions in the intermediate structures. Non-native interactions are found to be dominant in the unfolded state and also present in some intermediates, possibly hinder the folding process of the RNA.</p></div

Structures of the unfolded states.

<p>(A) Representative structures of the largest six clusters in the unfolded states. (B) HB map averaged over all the structures in the unfolded states. The formation probabilities are indicated by different colors, as quantified by the color scale beside the figure. The labels inside the figure are also colored, with yellow indicating tertiary interactions between H₁ and L₂ and white non-native HBs. (C) The number of bound metal ions and R_p as a function of Rg plotted for the largest six clusters.</p

Native structure of the RNA pseudoknot within gene32 mRNA of bacteriophage T2.

<p>(A) Secondary and tertiary structures of the RNA pseudoknot (PDB code: 2TPK). Two helices are labeled as Helix 1 (H₁) and Helix 2 (H₂) and are colored red and blue, respectively. Two loops are labeled as Loop 1 (L₁) and Loop 2 (L₂), and are colored orange and green, respectively. The same color code is used in all the figures unless otherwise indicated. (B) Surface model of a typical structure, viewed from two perpendicular directions, taken from a 200 ns MD simulation started from the native structure. Cations are plotted as yellow spheres. (C) Hydrogen bond (HB) map averaged over all the conformations in the same MD run. The formation probabilities are indicated by different colors, as quantified by the color scale beside the figure. The red labels indicate the HBs within two helices, and the yellow ones indicate that between H₂ and L₂, i.e., the tertiary interactions. (D) The fraction of the formed native HBs as a function of time in the same MD run.</p

Multiple folding mechanisms proposed for the RNA pseudoknot.

<p>The typical pathways are labeled as pathway-I, pathway-II, pathway-III and pathway-IV, and are colored by red, violet, blue and wine, respectively.</p

Representative structures of the intermediates.

<p>(A)-(F) are for the intermediates from I₁ to I₆, respectively. The nucleotides and metal ions are represented in the same way as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129089#pone.0129089.g001" target="_blank">Fig 1</a>.</p

Average HB maps of the intermediates.

<p>(A)-(F) are for the intermediates from I₁ to I₆. Different colors of the HBs indicate different formation probabilities as quantified by the color sale on the top of the figure. The labels of the hydrogen bonds are also colored, with the red, white and yellow colors indicating the native, non-native, and tertiary HBs, respectively.</p

The hydrogen bond maps for the intermediates.

<p>(A)–(D) are for the intermediate-II, III, IV, and V, respectively. The formation probabilities shown here are averaged on all the structures collected from multiple conventional MD simulations. Their values are indicated by the color scales. The hydrogen bonds pointed by the red arrows are native ones that exist in the native structure, while those pointed by the white arrows are non-native ones.</p

The <i>syn/anti</i> patterns of the intermediates.

<p>The bases that have formed native hydrogen bonds in between are plotted side-by-side and in the same plane. The red squares denote the nucleotides with configurations, the blue the ; and the gradient color indicates a fluctuating configuration between and . The nucleotides indicated by arrows correspond to either fluctuating (with gradient color) or wrong <i>syn/anti</i> configurations. Here by wrong we mean that they retain a <i>syn/anti</i> configuration different from the native one. The details of the trajectories are given in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003562#pcbi.1003562.s011" target="_blank">Figure S11</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003562#pcbi.1003562.s012" target="_blank">S12</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003562#pcbi.1003562.s013" target="_blank">S13</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003562#pcbi.1003562.s014" target="_blank">S14</a>.</p

Atomistic Picture for the Folding Pathway of a Hybrid-1 Type Human Telomeric DNA G-quadruplex

<div><p>In this work we studied the folding process of the hybrid-1 type human telomeric DNA G-quadruplex with solvent and ions explicitly modeled. Enabled by the powerful bias-exchange metadynamics and large-scale conventional molecular dynamic simulations, the free energy landscape of this G-DNA was obtained for the first time and four folding intermediates were identified, including a triplex and a basically formed quadruplex. The simulations also provided atomistic pictures for the structures and cation binding patterns of the intermediates. The results showed that the structure formation and cation binding are cooperative and mutually supporting each other. The <i>syn/anti</i> reorientation dynamics of the intermediates was also investigated. It was found that the nucleotides usually take correct <i>syn/anti</i> configurations when they form native and stable hydrogen bonds with the others, while fluctuating between two configurations when they do not. Misfolded intermediates with wrong <i>syn/anti</i> configurations were observed in the early intermediates but not in the later ones. Based on the simulations, we also discussed the roles of the non-native interactions. Besides, the formation process of the parallel conformation in the first two G-repeats and the associated reversal loop were studied. Based on the above results, we proposed a folding pathway for the hybrid-1 type G-quadruplex with atomistic details, which is new and more complete compared with previous ones. The knowledge gained for this type of G-DNA may provide a general insight for the folding of the other G-quadruplexes.</p></div

The detailed tertiary structures of the intermediates.

<p>(A)–(D) are for the intermediate-II, III, IV, and V, respectively. The structures are taken from the largest cluster in the corresponding conventional MD trajectories. They are slightly different from that shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003562#pcbi-1003562-g001" target="_blank">Figure 1</a>, which are obtained from BEMD simulations. The non-native hydrogen bonds are plotted as blue dashed lines and pointed by blue arrows. The ions bound to DNA are shown as yellow spheres.</p