Pathway modulation, circular permutation and rapid RNA folding under kinetic control.

The thermodynamics and folding kinetics of a circularly permuted construct of the ribozyme from Bacillus subtilis RNase P are analyzed and compared with the folding properties of the wild-type ribozyme using optical spectroscopy and catalytic activity. The folding of the wild-type ribozyme is slow due to the rearrangement of kinetically trapped species containing misfolded structures. To test whether any misfolded structure arises from interactions between the two independently folding domains of the RNase P RNA, a circular permuted form was created where one of the two phosphodiester bonds connecting these domains is broken. This construct folds approximately 15-fold faster (t1/2 approximately nine seconds) than the wild-type ribozyme at 37 degreesC. While the complete folding of both domains is kinetically indistinguishable in the wild-type ribozyme, one domain folds much faster than the other domain in the circularly permuted construct. Hence, the major kinetic trap in the folding of the wild-type RNase P RNA involves interdomain interactions. This kinetic trap is avoidable at 37 degreesC in the circularly permuted RNA. However, at temperatures below 30 degreesC or when refolding begins from an equilibrium intermediate stabilized by submillimolar concentrations of Mg2+, a subpopulation containing an interdomain misfold still forms. These results indicate that the folding pathway of this large RNA is highly malleable and can be under kinetic control.