Multiple Folding Pathways for the P4-P6 RNA Domain
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- 作者：Scott K. Silverman ; Michael L. Deras ; Sarah A. Woodson ; Stephen A. Scaringe ; and Thomas R. Cech
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：October 10, 2000
- 年：2000
- 卷：39
- 期：40
- 页码：12465 - 12475
- 全文大小：207K
- 年卷期：v.39,no.40(October 10, 2000)
- ISSN：1520-4995
文摘
We recently described site-specific pyrene labeling of RNA to monitor Mg2+-dependentequilibrium formation of tertiary structure. Here we extend these studies to follow the folding kinetics ofthe 160-nucleotide P4-P6 domain of the Tetrahymena group I intron RNA, using stopped-flow fluorescencewith ~1 ms time resolution. Pyrene-labeled P4-P6 was prepared using a new phosphoramidite that allowshigh-yield automated synthesis of oligoribonucleotides with pyrene incorporated at a specific 2'-amino-2'-deoxyuridine residue. P4-P6 forms its higher-order tertiary structure rapidly, with kobs = 15-31 s-1(t1/2 
20-50 ms) at 35 
C and [Mg2+] 10 mM in Tris-borate (TB) buffer. The folding rate increasesstrongly with temperature from 4 to 45 C, demonstrating a large activation enthalpy 
H
26 kcal/mol;the activation entropy S is large and positive. In low ionic strength 10 mM sodium cacodylate bufferat 35 C, a slow (t1/2 1 s) folding component is also observed. The folding kinetics are both ionicstrength- and temperature-dependent; the slow phase vanishes upon increasing [Na+] in the cacodylatebuffer, and the kinetics switch completely from fast at 30 C to slow at 40 C. Using synchrotron hydroxylradical footprinting, we confirm that fluorescence monitors the same kinetic events as hydroxyl radicalcleavage, and we show that the previously reported slow P4-P6 folding kinetics apply only to low ionicstrength conditions. One model to explain the fast and slow folding kinetics postulates that some tertiaryinteractions are present even without Mg2+ in the initial state. The fast kinetic phase reflects folding thatis facilitated by these interactions, whereas the slow kinetics are observed when these interactions aredisrupted at lower ionic strength and higher temperature.
20-50 ms) at 35 C and [Mg2+] 10 mM in Tris-borate (TB) buffer. The folding rate increasesstrongly with temperature from 4 to 45 C, demonstrating a large activation enthalpy H 26 kcal/mol;the activation entropy S is large and positive. In low ionic strength 10 mM sodium cacodylate bufferat 35 C, a slow (t1/2 1 s) folding component is also observed. The folding kinetics are both ionicstrength- and temperature-dependent; the slow phase vanishes upon increasing [Na+] in the cacodylatebuffer, and the kinetics switch completely from fast at 30 C to slow at 40 C. Using synchrotron hydroxylradical footprinting, we confirm that fluorescence monitors the same kinetic events as hydroxyl radicalcleavage, and we show that the previously reported slow P4-P6 folding kinetics apply only to low ionicstrength conditions. One model to explain the fast and slow folding kinetics postulates that some tertiaryinteractions are present even without Mg2+ in the initial state. The fast kinetic phase reflects folding thatis facilitated by these interactions, whereas the slow kinetics are observed when these interactions aredisrupted at lower ionic strength and higher temperature.