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

Naoto Hori; Natalia A. Denesyuk; D. Thirumalai

In order to highlight the structural changes that occur as C and f are varied, we used, Q, the fraction of native HB interactions as an order parameter. The [C, f ] phase diagram (Fig. 6A ) calculated using the average Q is similar to the R g -phase diagram (Fig. 4E) , indicating the presence of three states (Fig. 6A ). Using Q, we can also quantitatively assess the contributions from different structural elements to the stability of the PK and correctly infer the order in which the structural elements of the PK rupture as f is increased. In order to determine the structural details of each state, we calculated the individual Qs for the two stems (Q S1 , Q S2 ) and the two loops (Q L1 Q L2 ) (Fig. 6B) . The dependence of Q S1 as a function of C and f shows that Stem 1 (S1) is extremely stable and remains intact at all salt concentrations, rupturing only at f ≈ 10 pN (Fig. 6B , upper left panel). In contrast, the upper right panel in Fig. 6B shows that Stem 2 (S2) is only stably folded above a moderate salt concentration (C ≈ 80 mM) and low f . The stability difference between S1 and S2 can be explained by the number of canonical G-C base pairs; S1 has five G-C base pairs, whereas only three such pairs are in S2. Consequently, S1 cannot be destabilized even at low C, but its rupture to produce extended states requires the use of mechanical force. Above C > 100 mM, the fraction of contacts associated with S2 and the two loops (Q S2 , Q L1 and Q L2 ) simultaneously increase. All the interactions are, however, still not completely formed (Q ≈ 0.8) even at the highest salt concentration, C = 1200 mM. In particular, the contacts involving L2, Q L2 ≈ 0.6, implying that tertiary interactions are only marginally stable at T = 50 • C.