Salt Effects on the Thermodynamics of a Frameshifting RNA Pseudoknot under Tension

Naoto Hori; Natalia A. Denesyuk; D. Thirumalai

(1) At a low temperature, T = 20 • C, the PK is completely folded (F state) including all the structural motifs, S1, S2, L1 and L2. The existence of F state is also confirmed by the presence of a minimum at R ee ≈ 3 nm ( Fig. 2A) and at R g ≈ 1.3 nm (Fig. 2B) . These values are similar to those obtained directly from the crystal structure (R native ee = 2.8 nm, R native g = 1.3 nm). (2) At T = 60 • C, the free energy profile for U L HB shows that some of the tertiary interactions, involving L1 and L2, are no longer stably formed (Fig. S1B ). On the other hand, G(R g ) shows two stable minima, indicating that there are two possible states (Fig. 2B) . In one state, both stems S1 and S2 are formed but tertiary interactions are absent, corresponding to the I1 state identified using UV absorbance and differential scanning calorimetry experiments (Fig. 3) [30, 31] . The other conformation, in which only S1 is formed, has been also observed in experiments and is referred to as the I2 state. It should be noted that the distribution of U S HB also has two minima at 60 • C (Fig. S1A) whereas G(R ee ) has only one minimum ( Fig. 2A). (3) At a still higher temperature T = 80 • C, G(R ee ) and G(R g ) each have a minimum with S1 stably formed. This is also reflected in Fig. S1 , which shows a minimum at U S HB ≈ −30 kcal/mol and a minimum at U S HB ≈ 0. Thus, completely unfolded conformations, U state, and the I2 state are populated. (4) At T = 110 • C, both U S HB and U L HB are 0 , indicating that only the U state exists. This is also reflected in G(R ee ) and G(R g ), which show that the PK is completely unfolded. In both profiles (Figs. 2A and 2B), the center of minimum is located at larger values than in the F state (R ee ≈ 5 nm and R g ≈ 2 nm).