A thermodynamic framework and cooperativity in the tertiary folding of a Mg2+-dependent ribozyme

The folding thermodynamics of the catalytic domain from the Bacillus subtilis RNase P RNA is analyzed using circular dichroism and fluorescence spectroscopies, hydroxyl radical protection, and catalytic activity. Folding of this 255-nucleotide ribozyme can be described with three populated species: unfolded (U), intermediate (I), and native (N) states. The U-to-I transition primarily involves secondary structure formation, whereas the I-to-N transition is dominated by tertiary structure formation. The I-to-N transition is highly cooperative as indicated by the coincidence of the four probes applied here. Two isothermal methods are used to determine the stability of the N state relative to the I state at 10 and 37 degrees C. The first method measures the extent of Mg2+-induced folding without urea or at constant urea concentrations. The second method measures the extent of urea-induced unfolding at constant Mg2+ concentrations. Via application of a cooperative binding analysis, the Mg2+ transition midpoint (K-Mg), the Hill constant (n), and the urea-dependent surface burial parameter (In value) determined by both methods are identical, indicating that they report the same, reversible folding event. Three conclusions can be drawn from these results. (i) The folding free energy of a Mg2+-dependent tertiary RNA structure can be described by the K-Mg and n parameters according to a cooperative Mg2+ binding model. (ii) The Hill constant for this tertiary RNA structure probably represents the differential number of Mg2+ ions bound in the I-to-N transition. (iii) Under physiological conditions, the stability of this large ribozyme is similar to that of small globular proteins.