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

Scott K. Silverman, Thomas R. Cech

Research output: Contribution to journalArticle

Abstract

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 kobs = 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 kobs 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 kobs depends on the Mg2+ concentration, and the initial slope of kobs 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.

Original languageEnglish (US)
Pages (from-to)161-166
Number of pages6
JournalRNA
Volume7
Issue number2
DOIs
StatePublished - Feb 2001

Keywords

  • Activation energy
  • Fluorescence
  • Mutant
  • Phi value
  • Pyrene
  • Tertiary folding

ASJC Scopus subject areas

  • Molecular Biology

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