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°C with [Mg²⁺] ≈ 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 ΔG‡ of ∼8-16 kcal/mol is calculated, where the large range for ΔG‡ arises from uncertainty in the pre-exponential factor relating k_obs and ΔG‡. 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 Φ = ΔΔG‡/ΔΔG°′ are ≈0, implying a folding transition state in which most of the native-state tertiary contacts are not yet formed (an early folding transition state). The k_obs depends on the Mg²⁺ concentration, and the initial slope of k_obs versus [Mg²⁺] suggests that only ∼1 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 ΔG‡ 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.