6998 Nucleic Acids Research, 2007, Vol. 35, No. 20 In the presence of a stem, the PMF as a function of the T-loop RMSD (with respect to the crystal conformer) and the T-loop Rg is signiﬁcantly diﬀerent from that of the T-loop in isolation. Multiple distinct minima are observed (Figure 2B). The ﬁrst minimum [observed at RMSD = 2.2 A˚, Rg = 6.9 A˚] corresponds to a relatively intact hairpin with native-like T-loops. In these con- formers, we notice that the U4-A8 trans W/H closing bp is retained as part of the UA-handle (Figure 1), while the U-turn sub-motif (G5-G6-A7) loses its characteristic hydrogen bond contacts between the 20 OH position of G5 and the N7 of A7. Both positions G6 and A7 of the U-turn tend to ﬂip outside the loop and become fully solvated (Figure 2B). In some cases, they remain stacked outside the loop. We found the U-turn in the context of the T-loop to be much more ﬂexible than within the context of a GNRA tetraloop, which is more restricted by internal base stacking contacts and may only allow its second nucleotide position (N) to ﬂip out (25,38). This is expected because the third position of the loop (R) is stacked between the second position (N) and fourth position (A) in the context of a GNRA tetraloop. On the other hand, within the T loop context, A7 is at 7 A˚ from the U4-A8 trans W/H closing bp and only stacks with G6. Thus, the ﬂexibility of the U-turn is highly context dependent. In the T-loop within natural structural contexts (i.e. in the presence of tertiary interactions), a nucleotide is often intercalated between the last position (R) of the U-turn and the UA-handle. The second minimum [observed at RMSD = 3.8 A˚ Rg = 7 A˚ ] corresponds to a more expanded T-loop in which the UA-handle in addition to the U-turn is lost. In the third minimum [observed at RMSD = 5.2 A˚ Rg = 7.1 A˚ ], the T-loop starts to resemble a random coil. Our results suggest that the hairpin alone does not ensure the conformational stability of the U-turn sub- motif. Tertiary interactions are probably required to stabilize the U-turn. On the other hand, the UA-handle motif signiﬁcantly stabilizes the U:A trans W/H bp through additional stacking and H-bond contacts with the apical WC bp of the stem (Verzemnieks and Jaeger, manuscript in preparation). In summary, we found that the conformation of the T-loop is highly dependent on the overall conformation of the hairpin and it is signiﬁcantly diﬀerent from the stacking dominated coil conformer adopted by T-loop in isolation. The UA-handle motif requires at least one bulging nucleotide (C9) at the 30 end of the conserved A8 to bridge the U:A trans W/H bp to its WC bp (Figure 1A). In UA- handle motifs from the ribosome, these bulging nucleo- tides are typically ﬂipped outward to participate in additional tertiary interactions with their surrounding context (Verzemnieks and Jaeger, manuscript in prepara- tion). We were not able to observe any ﬂipping motion in the bulge nucleotide C9 during the 164 ns standard molecular dynamics simulation at 300 K (data not shown here). However, we did isolate structures whose bulge nucleotide C9 has ﬂipped inward during the replica exchange simulation, hence the importance of using enhanced sampling techniques to probe RNA folding. Our data are consistent with the observation that additional stabilizing contacts are necessary for stabilizing the bulge nucleotide and preventing it from ﬂipping inward in the context of the ribosome. Free energy surface of the hairpin at 300 K and folding transition state To gain insight into the energy landscape and folding mechanism of the T-loop hairpin, we determined the PMF using a larger range of structural parameters. At 300 K, at least two major basins are found when plotting the PMF as a function of the following parameters: Rg, RMSD of hairpin (compared to crystal conformer), number of native hydrogen bond (NH) and number of non-native hydrogen bonds (NN) (Figure 3). The basin with low RMSD (low NN or high NH), corresponds to the native basin, while the non-native basin is character- ized by high RMSD (high NN or low NH). It is worth mentioning that when the PMF is plotted over a limited set of parameters, each local minimum shown on the PMF may correspond to an ensemble of structures that are quite diﬀerent from each other, highlighting the diﬃculty in representing the entire spectrum of structural hetero- geneity using a limited set of structural parameters. Nevertheless, we found the use of Rg versus RMSD as a good coordinate sets to distinguish native from non-native structures. When the PMF is plotted as a function of the RMSD versus Rg at 300 K (Figure 3), we found in addition to the deep native state minimum (N), a non- native basin containing several sub-basins corresponds to diﬀerent trapped states. The relative energies of both the native and non-native basin are similar, which indicates that both minima are populated at this temperature and that additional interactions with the environment might be necessary to further stabilize one conformer over another. The native basin [at RMSD = 2–3 A˚ , Rg = 9–9.5 A˚ , NN = 1–2, NH = 7–9] includes the crystal-like states (N). The crystal structure has 12 native hydrogen bonds: 3 belong to the U-turn sub-motif, 2 belong to the U:A trans W/H bp and 7 belong to the helical stem. Visual analysis shows that state N has mostly complete stem formation (seven native hydrogen bonds). Most often, state N also has well-formed U:A trans W/H bp (two additional native hydrogen bonds). On the other hand, State N has a ﬂexible U-turn and does not contain any of the three native hydrogen bonds associated with this sub-motif found in the crystal structure. State N is relatively compact due to its fully formed helical stem. We distinguish the non-native basin [at RMSD = 5–7 A˚ , Rg = 8.5–10 A˚ , NN = 2–10, NH = 0–1] into two classes of structures: intermediates (I), coil/unfolded (U). State I is dominated by structures that fold in a symmetrical fashion such that the base pairs in the stem are approximately aligned with each other. State I can be considered as the pre-zipping state. State I is almost always slightly more expanded than state N because the helical strands are not close enough to form hydrogen bonds. State U, on the other hand, often folds into a shape that does not resemble that of the native hairpin. State U is likely to require more extensive structural