Multiple Folding Pathways for the P4−P6 RNA Domain

Abstract

We recently described site-specific pyrene labeling of RNA to monitor Mg(2+)-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 approximately 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) approximately 20-50 ms) at 35 degrees C and [Mg(2+)] approximately 10 mM in Tris-borate (TB) buffer. The folding rate increases strongly with temperature from 4 to 45 degrees C, demonstrating a large activation enthalpy DeltaH(double dagger) approximately 26 kcal/mol; the activation entropy DeltaS(double dagger) is large and positive. In low ionic strength 10 mM sodium cacodylate buffer at 35 degrees C, a slow (t(1/2) approximately 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 degrees C to slow at 40 degrees 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 Mg(2+) 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.

Rapid compaction during RNA folding

“…(As described in Discussion, it has recently been shown that increased ionic strength increases the rate of tertiary structure formation in P4-P6; ref. 34).…”

“…4B). A recent study has shown that under higher ionic strength conditions, upon addition of Mg 2ϩ , the P4-P6 domain forms its native tertiary structure on the timescale of milliseconds, indicating that any compaction of P4-P6 must also occur at least this fast, at least under the high ionic strength conditions (34). As formation of the native structure requires the P5abc element within P4-P6 to bend over and onto the rest of the subdomain (Fig.…”

Single-molecule studies highlight conformational heterogeneity in the early folding steps of a large ribozyme

“…In the latter case, preexisting secondary structures influence pathway selection resulting in the formation of a kinetically trapped intermediate, whereas folding from the Mg 2ϩ -free condition indicates that formation of native tertiary structure outpaces the formation of the trap-producing secondary structures. Different initial conditions also have a major impact on the folding behavior of the Tetrahymena group I intron (26,39). The folding surface has been characterized as having channels and a multidimensional landscape (36), whose topography also involves an interplay and timing between secondary and tertiary structure.…”

Improving small-angle X-ray scattering data for structural analyses of the RNA world

“…Multiple folding pathways are available to an RNA during in vitro refolding (Treiber et al 1998;Silverman et al 2000). In some cases, a pathway may lead to a kinetically trapped non-native conformation, thus producing structural heterogeneities or refolding artifacts in the sample (Rook et al 1999).…”