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 degrees C with [Mg2+] approximately 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 approximately 8-16 kcal/mol is calculated, where the large range for deltaG(double dagger) arises from uncertainty in the pre-exponential factor relating k(obs) and delta G(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(o') are approximately 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 approximately 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 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.
Silverman
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Department of Chemistry and Biochemistry, University of Colorado at Boulder, 80309-0215, USA. scott@scs.uiuc.edu
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D000818:Animals; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D009154:Mutation; D009690:Nucleic Acid Conformation; D011721:Pyrenes / Q000737:chemistry; D012313:RNA / Q000032:analysis / Q000302:isolation & purification; D016337:RNA, Catalytic / Q000737:chemistry*; D016053:RNA, Protozoan / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013768:Tetrahymena / Q000235:genetics*; D013816:Thermodynamics
D011721:Pyrenes; D016337:RNA, Catalytic; D016053:RNA, Protozoan; D012313:RNA; D008274:Magnesium