Figure 2.

(a) The magnesium-dependent UV thermal melting profiles of tP5abc. The UV thermal melting curves of tP5abc were recorded on a Gilford 250 spectrometer with four cell compartments. Samples were heated to 90°C and cooled to 4°C several times at a rate of 0.1°C/min. The increased UV absorbance as temperature increases indicates the breaking of secondary and tertiary structure of the RNA. The melting temperature of tP5abc does not change with the RNA concentration, showing that the thermal melting event is unimolecular. (b) The magnesium-dependent gel mobility of tP5abc in nondenaturing PAGE. The higher mobility of the wild-type tP5abc in 2 mM Mg²⁺ and 10 mM Na⁺ indicates that it folds into a more compact conformation than A186U, a mutant that prevents the tertiary interactions. (c) One-dimensional imino NMR spectra of tP5abc wild-type and A186U. The wild-type molecule folds in 2 mM Mg²⁺, whereas the A186U mutant does not fold, as shown by the unchanged imino spectra up to 5 mM Mg²⁺. Six arrows point to the imino protons of the three G⋅U base pairs in the extended tP5abc that disappear on tertiary folding. *, the imino proton of G169 within the GCAA tetraloop that also disappears when P5c rearranges. In contrast, imino proton resonances of the three G⋅U base pairs of A186U do not change in the presence of Mg²⁺, indicating no change in the secondary structure.