We recently described site-specific pyrene labeling of RNA to monitor Mg2+-dependent equilibrium formation of tertiary structure. Here we extend these studies to follow the folding kinetics of the 160-nucleotide P4-P6 domain of the Tetrahymena group I intron RNA, using stopped-flow fluorescence with ~1 ms time resolution. Pyrene-labeled P4-P6 was prepared using a new phosphoramidite that allows high-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 k(obs) = 15-31 s-1 (t(1/2) ≃ 20-50 ms) at 35 °C and [Mg2+] ≃ 10 mM in Tris-borate (TB) buffer. The folding rate increases strongly with temperature from 4 to 45 °C, demonstrating a large activation enthalpy ΔH((+)) ≃ 26 kcal/mol; the activation entropy ΔS((+)) is large and positive. In low ionic strength 10 mM sodium cacodylate buffer at 35 °C, a slow (t(1/2) ≃ 1 s) folding component is also observed. The folding kinetics are both ionic strength- and temperature-dependent; the slow phase vanishes upon increasing [Na+] in the cacodylate buffer, and the kinetics switch completely from fast at 30 °C to slow at 40 °C. Using synchrotron hydroxyl radical footprinting, we confirm that fluorescence monitors the same kinetic events as hydroxyl radical cleavage, and we show that the previously reported slow P4-P6 folding kinetics apply only to low ionic strength conditions. One model to explain the fast and slow folding kinetics postulates that some tertiary interactions are present even without Mg2+ in the initial state. The fast kinetic phase reflects folding that is facilitated by these interactions, whereas the slow kinetics are observed when these interactions are disrupted at lower ionic strength and higher temperature.
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