SHAPE-restrained secondary structure model for free STMV RNA.

<p>Nucleotides are colored according to their SHAPE reactivity (see scale). Inset shows a box plot comparing the distribution of SHAPE reactivity values between base paired and single-stranded nucleotides. Each grey box represents the interquartile range (IQR) of the data; the bottom and top edges of the box are the 25th and 75th percentiles, respectively. The band near the middle of each box is the median value. The whiskers above and below each box extend to the most extreme data points not considered outliers. Outliers are plotted individually as crosses. Points are outliers if they are greater than 1.5 IQR from the 75th percentile or less than 1.5 IQR from the 25th percentile. In this secondary structure model, the distribution for base paired nucleotides is narrower and has a much lower median value than the distribution for single-stranded nucleotides.</p

Distribution of double-helical RNA segments in the STMV virion.

<p>The crystal structure of STMV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054384#pone.0054384-Larson1" target="_blank">[4]</a> reveals 30 segments of double-helical RNA (blue). Each helix contains 9 base pairs, centered on a crystallographic two-fold axis. An icosahedral cage (pink) is shown for reference. Adopted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054384#pone.0054384-Zeng1" target="_blank">[8]</a>.</p

Identification of possible double-helical stems corresponding to those seen in the crystal structure.

<p>There are 30 stems in the crystal structure, each containing nine base pairs with an additional base stacked at each 3′ end, <i>i.e.</i>, 20 nucleotides (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054384#pone-0054384-g001" target="_blank">Figure 1</a>). A model that connects successive stems would require something on the order of 5–10 nucleotides per connection. This figure shows how our secondary structure model might be organized to fit into the STMV capsid, with a sufficient number of stems to cover the 30 edges of the icosahedral frame, as required by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054384#pone-0054384-g001" target="_blank">Figure 1</a>.</p

Predicted secondary structure at the 3′ end of STMV RNA.

<p>Secondary structure for the 406 3′-terminal nucleotides of STMV RNA proposed by Gultyaev <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054384#pone.0054384-Gultyaev1" target="_blank">[10]</a>. Nucleotides are colored according to their SHAPE reactivity (see scale). The SHAPE data supports the tRNA-like structure and the five stem-loops (nucleotides 728–1058), but does not support the second pseudoknot domain (nucleotides 653–727).</p

Schroeder secondary structure model for encapsidated STMV RNA.

<p>Schroeder <i>et al.</i> predicted this secondary structure on the basis of the co-replicational folding and assembly hypothesis, along with chemical probing data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054384#pone.0054384-Schroeder1" target="_blank">[7]</a>. Nucleotides are colored according to their SHAPE reactivity (see scale), and the hairpin loops are numbered from 1 to 30. Hairpins 1, 3, 10–13, 17, 21–22, and 25 are clearly inconsistent with the SHAPE data.</p

Effect of Mg²⁺ on the SHAPE reactivity profile of free STMV RNA.

<p>SHAPE reactivities for STMV RNA in the presence (top) and absence (middle) of Mg²⁺. The difference plot (bottom) shows that 10 mM Mg²⁺ has little effect on the SHAPE reactivity profile.</p

SHAPE-restrained secondary structure of free STMV RNA with a tRNA-like fold at the 3′ end.

<p>This alternate model of the STMV RNA was obtained by combining the SHAPE-restrained secondary structure predicted separately for nucleotides 1–727 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054384#pone-0054384-g002" target="_blank">Figure 2</a>) with the Gultyaev <i>et al.</i> prediction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054384#pone.0054384-Gultyaev1" target="_blank">[10]</a> for nucleotides 728–1058 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054384#pone-0054384-g005" target="_blank">Figure 5</a>). Nucleotides are colored according to their SHAPE reactivity (see scale). The extended central domain (nucleotides 64–720) is identical to that of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054384#pone-0054384-g002" target="_blank">Figure 2</a>.</p

Minimum free energy (MFE) structure obtained for STMV RNA without the SHAPE data.

<p>The structure was predicted using RNAstructure with default parameters. Nucleotides are colored according to their SHAPE reactivity (see scale). The SHAPE data are not consistent with this model, since several base paired regions have high reactivity values.</p

Mapping the chemical probing data from Schroeder <i>et al.</i>[7] onto the SHAPE-restrained secondary structure of <i>in vitro</i> transcribed STMV RNA.

<p>Red circles indicate nucleotides modified by DMS, kethoxal, or CMCT. The data do not appear to clearly rule out the proposed secondary structure of residues 1–730. A substantial number of the modifications occur in predicted loops, bulges, and single-stranded regions (67 out of 119 hits). Many of the reactive base-paired nucleotides are in A-U or G-U base pairs immediately adjacent to a predicted bulge loop (<i>e.g.</i>, 128, 185, 187, 192, 213, 413–414, 556, 561, 652–653, 663, 675), while others (382–390 and 503–515) are in a predicted stem that has two bulges and has no run of more than three consecutive base pairs, so it should be prone to fraying.</p

RNA Folding and Catalysis Mediated by Iron (II)

<div><p>Mg²⁺ shares a distinctive relationship with RNA, playing important and specific roles in the folding and function of essentially all large RNAs. Here we use theory and experiment to evaluate Fe²⁺ in the absence of free oxygen as a replacement for Mg²⁺ in RNA folding and catalysis. We describe both quantum mechanical calculations and experiments that suggest that the roles of Mg²⁺ in RNA folding and function can indeed be served by Fe²⁺. The results of quantum mechanical calculations show that the geometry of coordination of Fe²⁺ by RNA phosphates is similar to that of Mg²⁺. Chemical footprinting experiments suggest that the conformation of the <em>Tetrahymena thermophila</em> Group I intron P4–P6 domain RNA is conserved between complexes with Fe²⁺ or Mg²⁺. The catalytic activities of both the L1 ribozyme ligase, obtained previously by <em>in vitro</em> selection in the presence of Mg²⁺, and the hammerhead ribozyme are enhanced in the presence of Fe²⁺ compared to Mg²⁺. All chemical footprinting and ribozyme assays in the presence of Fe²⁺ were performed under anaerobic conditions. The primary motivation of this work is to understand RNA in plausible early earth conditions. Life originated during the early Archean Eon, characterized by a non-oxidative atmosphere and abundant soluble Fe²⁺. The combined biochemical and paleogeological data are consistent with a role for Fe²⁺ in an RNA World. RNA and Fe²⁺ could, in principle, support an array of RNA structures and catalytic functions more diverse than RNA with Mg²⁺ alone.</p> </div