An early transition state for folding of the P4-P6 RNA domain

Tertiary folding of the 160-nt P4-P6 domain of the Tetrahymena group I intron RNA involves burying of substantial surface area, providing a model for the folding of other large RNA domains involved in catalysis, Stopped-flow fluorescence was used to monitor the Mg2+-induced tertiary folding of pyrene-labeled P4-P6. At 35 degreesC with [Mg2+] approximate to 10 mM, P4-P6 folds on the tens of milliseconds timescale with k(obs) = 15-31 S-1. From these values, an activation free energy DeltaG(double dagger) of similar to8-16 kcal/mol is calculated, where the large range for DeltaG(double dagger) arises from uncertainty in the preexponential factor relating k(obs) and DeltaG(double dagger). The folding rates of six mutant P4-P6 RNAs were measured and found to be similar to that of the wild-type RNA, in spite of significant thermodynamic destabilization or stabilization. The ratios of the kinetic and thermodynamic free energy changes Phi = Delta DeltaG(double dagger)/Delta DeltaG degrees' are approximate to 0 implying a folding transition state in which most of the native-state tertiary contacts are not yet formed tan early folding transition state). The kobs depends on the Mg2+ concentration, and the initial slope of k(obs) versus [Mg2+] suggests that only similar to1 Mg2+ ion is bound in the rate-limiting folding step. This is consistent with an early folding transition state, because folded P4-P6 binds many Mg2+ ions. The observation of a substantial DeltaG(double dagger) despite an early folding transition state suggests that a simple two-state folding diagram for Mg2+-induced P4-P6 folding is incomplete, Our kinetic data are some of the first to provide quantitative values for an activation barrier and location of a transition state for tertiary folding of an RNA domain.