Nucleobases Undergo Dynamic Rearrangements during RNA Tertiary Folding

Abstract

The tertiary structure of the GTPase center (GAC) of 23S ribosomal RNA (rRNA) as seen in cocrystals is extremely compact. It is stabilized by long-range hydrogen bonds and nucleobase stacking and by a triloop that forms within its three-way junction. Its folding pathway from secondary structure to tertiary structure has not been previously observed, but it was shown to require Mg²⁺ ions in equilibrium experiments. The fluorescent nucleotide 2-aminopurine was substituted at selected sites within the 60-nt GAC. Fluorescence intensity changes upon addition of MgCl₂ were monitored over a time-course from 1ms to 100s as the RNA folds. The folding pathway is revealed here to be hierarchical through several intermediates. Observation of the nucleobases during folding provides a new perspective on the process and the pathway, revealing the dynamics of nucleobase conformational exchange during the folding transitions.

Keywords: 2-aminopurine fluorescence; GTPase center RNA; RNA folding kinetics; stopped-flow fluorescence.

Figure 1

A) Secondary structure of the E. coli GAC U1061A, with phylogenetically conserved bases in magenta and tertiary contacts observed in the cocrystals indicated by lines. Numbered nucleotides were substituted with 2-aminopurine. B) Sequences of hairpin constructs with sites of 2AP substitution in green. C) Cocrystal structure (1HC8) of E. coli GAC with 2AP substitution sites in green.

Figure 2

Mg²⁺ dependence of 2AP-GAC stability. A. First derivative of absorbance (A₂₆₀) vs temperature (dA/dT) which reveals the conformational transitions of the RNAs. Melting was measured in 100 mM KCl, 3 mM MgCl₂, 10 mM sodium cacodylate, pH 6.5. The lower temperature transition is assigned to tertiary structure melting; the second transition is secondary structure melting. B. MgCl₂ titration of 2AP fluorescence at 20°C. [GAC] = 2 μM in 100 mM KCl, 10 mM sodium cacodylate, pH 6.5.

Figure 3

Stopped flow fluorescence traces of 2AP-GAC at 20° C. Top row from left to right A1061AP, A1067AP, A1069AP. Bottom row from left to right A1070AP, A1089AP, A1095AP. MgCl₂ additions from 3 (blue), 5, 8, 20, 40, 80, to 100 (red) mM in 100 mM KCl, 10 mM sodium cacodylate. The black trace is buffer-only mixing control.

Figure 4

Relaxation times and corresponding observed rates (1/τ) from global fits. Errors are contained within the data points. The scatter in the first points in the top plot is due to sparse sampling. An example of global fitting of stopped-flow traces for all 2AP-GAC molecules is shown for addition of 20 mM MgCl₂. Black traces are data; red lines are fit. All progress curves and fits are shown in Supplemental Figures 2 and 3.

Figure 5

Stopped-flow fluorescence traces of the A1095AP U-turn hairpin construct. Black trace is control addition of buffer only. Adding MgCl₂ to 3, 5, 8, 20, 80,100 mM results in an immediate increase in fluorescence intensity but no subsequent transitions. Experiments with other hairpins showed similar rapid intensity changes. [RNA] = 100 nM in 100 mM KCl, 10 mM sodium cacodylate pH 6.5. 20° C.

Figure 6

Visualization of a possible folding scheme. The first structure is the starting secondary structure. The second is formed in < 1.5 ms and illustrates electrostatic relaxation upon addition of Mg²⁺ ions. The third structure is the counterion collapsed state. In the fourth structure, the T+loop structure is changed when specific Mg²⁺ ions associate, leading to a state where the second ion binding occurs when the hairpins interact. Finally, the last state is the tertiary structure.

Figure 7

Backbone trace of human[59] (pink) and Thermus[60] (green) GACs from ribosome crystal structures. Most of the variation is in the T+loop and the junction phosphodiester backbone. Thermus ribosome lacked L11, human ribosome includes L12 (equivalent to L11) bound to the GAC.