Multiple folding pathways for the P4-P6 RNA domain

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 similar to 1 ms time resolution. Pyrene-labeled P4-P6 was prepared using a new phosphoramidite that allows high-yield automated synthesis of oligorib onucleotides with pyrene incorporated at a specific 2'-amino-2'-deoxyuridin e residue. P4-P6 forms its higher-order tertiary structure rapidly, with k( obs) = 15-31 s(-1) (t(1/2) approximate to 20-50 ms) at 35 degrees C and [Mg 2+] approximate to 10 mM in Tris-borate (TB) buffer. The folding rate incre ases strongly with temperature from 4 to 45 degrees C, demonstrating a larg e activation enthalpy Delta H-not equal approximate to 26 kcal/mol; the act ivation entropy Delta S-not equal is large and positive. Tn low ionic stren gth 10 mM sodium cacodylate buffer at 35 degrees C, a slow (t(1/2) x 1 s) f olding component is also observed. The folding kinetics are both ionic stre ngth- and temperature-dependent; the slow phase vanishes upon increasing [N a+] in the cacodylate buffer, and the kinetics switch completely from fast at 30 degrees C to slow at 40 degrees C. Using synchrotron hydroxyl radical footprinting, we confirm that fluorescence monitors the same kinetic event s as hydroxyl radical cleavage, and we show that the previously reported sl ow P4-P6 folding kinetics apply only to low ionic strength conditions. One model to explain the fast and slow folding kinetics postulates that some te rtiary interactions are present even without Mg2+ in the initial state. The fast kinetic phase reflects folding that is facilitated by these interacti ons, whereas the slow kinetics are observed when these interactions are dis rupted at lower ionic strength and higher temperature.