INTERMEDIATES AND KINETIC TRAPS IN THE FOLDING OF A LARGE RIBOZYME REVEALED BY CIRCULAR-DICHROISM AND UV ABSORBENCY SPECTROSCOPIES AND CATALYTIC ACTIVITY

The folding thermodynamics and kinetics for the ribozyme from Bacillus subtilis RNase P are analyzed using circular dichroism and UV absorba nce spectroscopies and catalytic activity. At 37 degrees C, the additi on of Mg2+ (K-d similar to 50 mu M) to the unfolded state produces an intermediate state within 1 ms which contains a comparable amount of s econdary structure as the native ribozyme. The subsequent transition t o the native state (K(d)([Mg])similar to 0.8 mM, Hill coefficient simi lar to 3.5) has a half-life of hundreds of seconds as measured by circ ular dichroism at 278 nm and by a ribozyme activity assay. Surprisingl y, the formation of the native structure is accelerated strongly by th e addition of a denaturant; similar to 30-fold at 4.5 M urea. Thus, th e rate-limiting step entails the disruption of a considerable number o f interactions. The folding of this, and presumably other large RNAs, is slow due to the structural rearrangement of kinetically trapped spe cies. Taken together with previous submillisecond relaxation kinetics of tRNA tertiary structure, we suggest that error-free RNA folding can be on the order of milliseconds.