6996 Nucleic Acids Research, 2007, Vol. 35, No. 20 address the eﬀects of sequence and structural parameters on hairpin folding (42). The T-loop motif was ﬁrst observed in tRNAs and later found in viral RNAs, tmRNAs and ribosomal RNA (16). It is an important structural building block involved in formation of long-range tertiary interactions (16). The original deﬁni- tion of the T-loop included as key components: a U-turn motif, a UA trans Watson–Crick/Hoogsteen (W/H) bp stacked on a Watson–Crick bp and several bulging residues 30 to the conserved adenosine (16). It was later discovered that the T-loop motif could also exist in single strand non-ordered regions without requiring the U:A trans W/H bp to be stacked on a WC bp (20). The deﬁnition of the T-loop motif, as a 5-nt loop formed of a U-turn and a UA trans W/H closing bp (20), was thus adopted in this article. Precisely how each key component of the T-loop contributes to the stability, folding and function of this motif remains poorly understood. In this study, we present all-atom explicit solvent simulations of the T-loop using an enhanced sampling technique known as replica exchange molecular dynamics simulations (REMD) (43–45). The T-loop studied here consists of the U-turn sub-motif often found in the GNRA tetraloop, the U-A trans W/H closing bp, and a single nucleotide bulge at the 30 end of the conserved adenine (Figure 1). The hairpin includes the T-loop and a 3-bp long stem. When the U:A trans W/H bp is stacked on a WC bp in the presence of a hairpin stem, a new motif called the UA-handle is created (Verzemnieks and Jaeger, manuscript in preparation). Consequently, our model system can be decomposed into many diﬀerent subunits/ components and it represents a minimal RNA structure Figure 1. T-loop RNA hairpin with its components: (A) sequence signature of the U-turn sub-motif, the U:A trans W/H bp, the UA-handle and the T-loop. The UA-handle motif comprises at least 5 nt that form a U:A trans W/H bp, a WC pb and a bulge. N stands for any nucleotides (G, A, U or C); R stands for purine (G or A); Y stands for pyrimidine (U or C). (B) Sequence of the motifs simulated in this study, which includes the U-turn motif, the closing U:A trans W/H bp, the single nucleotide C in bulge, the UA-handle and the hairpin stem. (C) 3D structures of the corresponding model systems in their crystal conformation. The trans W/H base pair is indicated according to the nomenclature of base pairs deﬁned by Leontis and Westhof (60). with realistic hierarchical organization that is amenable to computational studies (Figure 1). Our interests here are to (i) compare the behavior a sub-motif (T-loop) with and without a local context (the stem), (ii) address how and when the sequence information in the T-loop is used to guide RNA folding, (iii) compare the mechanism by which the T-loop hairpin folds with that of the tetraloop hairpins (that share the U-turn submotif and the non-Watson–Crick closing base pair with the T-loop hairpins) and (iv) probe the energy landscape for folding of the RNA hairpin. METHODS AND MODELS Models and sequences Simulations were performed on two molecules: a T-loop in isolation and a hairpin containing the T-loop (see Figure 1). The T-loop in isolation has sequence 50-UGGAAC-30. The hairpin containing the T-loop has sequence 50-UCU-[UGGAAC]-AGA-30. Starting struc- tures for both molecules were dissected from the crystal structure of Haloarcula marismortui large ribosomal subunit (Residues 310–321, PDB:1JJ2) (33,46). Replica exchange molecular dynamics (REMD) simulation of T-loop in isolation and as part of a hairpin Replica exchange molecular dynamics (REMD) simula- tions were performed on the isolated T-loop and on the hairpin systems described above using the Sander module from the Amber 8 package (47) and the Amber parm99 force ﬁeld (48). The systems were neutralized by sodium counter-ions and solvated in explicit TIP3P water (49) using the LeaP module. A truncated octahedron water box was used such that the RNA starting structure is no less than 10 A˚ from the edge of the water box. Hydrogen-containing bonds were constrained by the SHAKE algorithm (50). Long-range electrostatic interac- tions were treated using the Particle Mesh Ewald method (PME) (51), and non-bonded cutoﬀs of 10 A˚ were used in the simulation. The system was minimized using the steepest-descent (500 steps) followed by the conjugate gradient (500 steps) method. Equilibration was per- formed under constant temperature and pressure of 1 atm. Production runs were carried out under constant temperature and volume. Constant temperature or con- stant pressure is achieved using the weak-coupling algorithm (52). The integration time-step is 2 fs. The simulation of the T-loop in isolation involved 28 replicas at 28 diﬀerent temperatures ranging from 298 to 400 K. Each replica of the T-loop in isolation was simulated for 20 ns, with a cumulative simulation time of 560 ns. The hairpin simulation involved 30 replicas at 30 diﬀerent temperatures ranging from 298 to 400 K. Each replica of the hairpin was simulated for 80 ns, with a cumulative simulation time of 2.4 ms. Temperatures were exponentially spaced. The acceptance ratio for replica exchange is within the range of 10–20%. The starting structure for the T-loop in isolation is a coil-like structure obtained from a standard MD simulation at 300 K. The hairpin simulations were initiated from two diﬀerent