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 language | English (US) |
|---|---|
| Pages (from-to) | 161-166 |
| Number of pages | 6 |
| Journal | RNA |
| Volume | 7 |
| Issue number | 2 |
| DOIs | |
| State | Published - Feb 1 2001 |
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Keywords
- Activation energy
- Fluorescence
- Mutant
- Phi value
- Pyrene
- Tertiary folding
ASJC Scopus subject areas
- Molecular Biology
Cite this
An early transition state for folding of the P4-P6 RNA domain. / Silverman, Scott K.; Cech, Thomas R.
In: RNA, Vol. 7, No. 2, 01.02.2001, p. 161-166.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - An early transition state for folding of the P4-P6 RNA domain
AU - Silverman, Scott K.
AU - Cech, Thomas R.
PY - 2001/2/1
Y1 - 2001/2/1
N2 - 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.
AB - 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.
KW - Activation energy
KW - Fluorescence
KW - Mutant
KW - Phi value
KW - Pyrene
KW - Tertiary folding
UR - http://www.scopus.com/inward/record.url?scp=0035261219&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0035261219&partnerID=8YFLogxK
U2 - 10.1017/S1355838201001716
DO - 10.1017/S1355838201001716
M3 - Article
C2 - 11233973
AN - SCOPUS:0035261219
VL - 7
SP - 161
EP - 166
JO - RNA
JF - RNA
SN - 1355-8382
IS - 2
ER -