Hidden Structural Modules in a Cooperative RNA Folding Transition. Gracia et al

Quantitative single nucleotide resolution RNA footprinting methods probe structural accessibility at each nucleotide in the RNA structure, i.e. high reactivity indicates unpaired/flexible positions and low reactivity base-paired/rigid positions. By steadily increasing the concentration of free Mg2+, we induce RNA folding and observe the structural transitions. We quantify the Mg2+-dependent changes in reactivity by fitting the observed transitions to a sigmoidal binding equation. We extract the transition midpoint parameter from the fit allowing for determination of the Mg2+ requirement for folding at a given nucleotide (i.e. the concentration required to reach half maximal saturation). The spreadsheets contain nucleotide reactivity's starting and ending with the 5' and 3' hairpin loop, respectively, (GAGUA) of the cassette. The conclusion from the data for WT P5abc was that there are multiple Mg2+-dependent transitions involving structural modules. To test for cooperativity between two of these modules, P5c and the metal core, we performed footprinting experiments using several mutants that either blocked the transition in P5c (U167C or U167C/U177C) or blocked folding of the metal core (A186U or ArichU). A lack of signal change at the mutated region (for example, U168 for mutant U167C) indicates that the module of interest is blocked by the mutation. We observed Mg2+-dependent changes in the unmutated region similar to WT, but the Mg2+ was greater than WT, consistent with cooperativity between the structural modules. We designed mutations that preformed the P5c module (Nat+3) and found that the overall Mg2+ requirement for folding the structure was significantly reduced compared to WT (e.g. nucleotides U135, A139, G163, G164, A186). We also partially restored folding of the blocked mutants by inserting restoration nucleotides that replace the blocked region (U167C_U177C_G_GUins). This restoration mutant decreased the Mg2+ requirement and restored signals associated with folding to WT levels compared with the U167C blocking mutant (e.g. nucleotides U135, A139, G163, GinsG164, GinsG176, A186). We utilized mutations that block the long-range interactions that form between the folded P5c and metal core modules (U168C) and observed that folding of the modules was not perturbed compared to WT (e.g. U135, A139, G163, A186). At concentrations above 10 mM Mg2+, we observed that some positions were perturbed for folding compared to WT (e.g. A184 SHAPE remains moderately reactive in U168C at high Mg2+ concentrations). We used mutations that preform the P5c module (C165A_G175U) to observe the formation of long range interactions between the folded modules. We found that signals associated with the P5c packing interaction (e.g. A173 DMS) were destabilized when the metal core module was blocked (C165A_G175U_A186U) compared to WT, consistent with cooperativity between the structural modules during formation of long-range interactions.