The structure, folding kinetics, and dynamics of long poly(UG) RNA

Figures

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  Figure 1.. (A) Structure of the (GU)12 pUG fold (PDB: 8TNS, 7MKT). The left-handed G4 is comprised of four segments, each with 3 GU repeats. The segments are color coded from 5′ to 3′ as red, cyan, green, and blue. Three central potassium ions are shown in purple. (B) The G1-quartet. (C) Schematic of the pUG fold topology, with G and U nucleotides shown as filled and open circles, respectively. (D) Short-hand description of the pUG-fold. Upper case nucleotides are Anti, and lower case are Syn. Bold nucleotides are 2′-endo and italicized nucleotides are 3′-endo. Inverted nucleotides indicate inverted strand polarity relative to the 5′ nucleotide. Small Us represent bulged or looped nucleotides..

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  Figure 2.. (A) CD spectra of (GU)12, (GU)13, (GU)15, (GU)18, and (GU)29. (B) Plot of fraction folded vs nucleotides in a pUG fold per total nucleotides, (pUG nt)/(RNA nt). The fraction folded is measured as the normalized magnitude of the CD signals relative to (GU)12, (Δϵ(RNA)/Δϵ((GU)12)*0.95) at 245 nm (circle), 281 nm (triangle), and 304 nm (square). Two points for (GU)29 depict a folding ratio for 1 pUG fold (filled markers) or 2 pUG folds (open markers).

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  Figure 3.. Nuclease mapping of (GU)29 using RNase T1. The first lane contains markers for (GU)12 and (GU)24 RNAs, while the second lane contains untreated (GU)29 in K+ buffer. RNase treatments are indicated. The three possible RNase resistant fragments are indicated and correspond to 5′ hydroxyl (HO-), 5′ triphosphate (PPP), 3′ hydroxyl, and 3′ cyclic phosphate (>P) termini containing cleavage products.

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  Figure 4.. (A) Native PAGE analysis of pUG RNAs of various lengths in K+ or Li+. (B) Plot of distance migrated in mm versus the number of nucleotides showing linear relationship (R2= 0.98) for single stranded pUG RNAs (Li+) and single pUG fold bands (K+). Double pUG fold outliers differentiated with open squares. (A/B) Single pUG folds and double pUG folds are marked with one or two asterisks, respectively.

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  Figure 5.. Double pUG fold model of (GU)24. Gs are green, Us are blue, and potassium ions are purple. 5′ and 3′ ends are highlighted in red. (A) depicts the initial conformation following energy minimization and (B) depicts the stacked conformation observed from 486 ns to 3 μs of MD simulation.

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  Figure 6.. HDX NMR of (GU)12. (A) HDX mechanism where H-bonded protons in quartets, (H)quartet, must open, (H)open, to allow the proton to be exchanged, (H)exchanged, with deuterium. (B) 1D 1H NMR showing loss of imino proton signal at given times following transfer into D2O. (C) Exponential decay of the normalized peak integrals (I(t)/I(0)), over time. (D) Summary of average HDX rates (kex) and quartet half-lives for the G3 and G5 quartets, G23 was measured separately.

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  Figure 7.. Biphasic HDX of (GU)13, (GU)15, and (GU)18 compared to (GU)12. (A) Plot of loss of imino proton signal measured as normalized integrals (I(t)/I(0)) of the entire imino region over time fit to an exponential decay for (GU)12 and biexponential decays for (GU)13, (GU)15, and (GU)18. (B) Zoomed in plot of boxed region from (A) highlighting the fast phase of HDX biexponential decay for (GU)13, (GU)15, and (GU)18.

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  Figure 8.. 1D 1H NMR of (GU)12, (GU)13, (GU)15, (GU)18, and the 50 nt sequence (GU)12AU(GU)12 from the HO-1 gene. Dotted lines mark chemical shifts of the (GU)12 pUG fold.

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  Figure 9.. Schematic models of pUG fold dynamics. (A) Segmental register exchange of an internal pUG fold in (GU)15 involving flanking 5′ and 3′ segments. Gs shown in filled circles and Us shown as open circles. Single stranded nucleotides (yellow and orange) exchange into the structure through the formation of expanded bulges or propeller loops. (B) Global structural rearrangement of (GU)29 resulting in an increase in double pUG folds.