Nucleobases Undergo Dynamic Rearrangements during RNA Tertiary Folding

•Mg2 +-dependent RNA folding pathways reveal how tertiary structures utilize ions.•GAC tertiary folding pathway has four intermediates; two require Mg2 +.•GAC folding kinetics range from milliseconds (local) to seconds (global).•Fluorescent bases reveal transient conformational changes before global folding.

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 1 ms to 100 s 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.

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