2880 Nucleic Acids Research, 2006, Vol. 34, No. 10 4096 data points in t2 and 512 increments in t1. The data were apodized with a shifted sine-bell square function in both dimensions and processed to a 4K · 1K matrix. Both 31P-decoupled (on resonance, continuous decoupling) and 31P-coupled spectra were recorded under the same conditions. For the TOCSY experiment, a clean DIPSI-2rc (21) was used, with a 90 pulse of 18.2 ms and a mixing tune of 65 ms. The total TOCSY mixing time was set to 64 ms. The spectrum was acquired with 32 scans, 2048 data points in t2 and 512 FIDs in t1. The data were apodized with a shifted sine-bell square function in both dimensions and processed to a 2K · 1K matrix. The NOESY experiments were acquired with mixing times of 50, 100, 150, 250 and 300 ms, 32 scans, 2048 data points in t2 and 512 increments in t1. A 1H-31P HETCOR (22) spectrum was acquired with 256 scans, 2048 data points in the proton dimension, t2 and 256 increments in the phosphorus dimension, t1, over sweep widths of 4200 and 1600 Hz, respectively. The data were apodized with a shifted sine-bell square function in both dimensions and processed to a 2K · 1K matrix. A natural abundance 1H,13C HSQC was recorded with sensitivity enhancement and gradient coherence selection optimized for selection of CH2 groups (23) using 72 scans and 256/512 complex data points and 10 000/12 500 Hz spectral widths in t1 and t2, respectively. Titration with MgCl2 was monitored by 2D natural abundance [1H,13C]-HSQC spectra, 1D 1H and 31P spectra. NMR derived restraints Distance restraints were derived from NOESY spectra recor- ded with 50, 100 and 150 ms mixing times, using the FELIX 97.00 software (Accelrys ). Based on the build-up curves, inter-proton distances were calculated. An experimental error (±20%) was used on the calculated inter-proton dis- tances. The calibration of NOE cross peak intensities was done against the H5–H6 cross peaks as an internal standard and resulted in 280 restraints. Sugar puckers of the ribose’s were inferred from the weak H10 to H20 scalar couplings. Residues C28–C32, A38–G42 and 6 t A37 have H10 to H20 scalar couplings varying from 0 and 2 Hz and therefore were restrained to the N pucker con- formation (24) [dihedral restraints applied: H10-C10-C20-H20 (99.2 ± 20 ), H20-C20-C30-C30 (39.4 ± 20 ), H30-C30-C40-H40 ( 162.0 ± 20 )]. Residues U27, U33–U36 and A43 have lar- ger H10 to H20 couplings (between 3 and 7 Hz). For this reason no restraints were applied on the sugar rings of these residues during the structure calculation. The normal 31P chemical shifts of most residues were used to restrain a and z torsion angles (0 ± 120 ) (24). The a of residue U33 and z torsion angle of residue C32 were not res- trained since U33:P showed a chemical shift deviating from the typical values of A-form RNA. Except for residue U36, nicely resolved H40-P(n)-crosspeaks in the 2D 1H-detected [1H,31P]HETCOR, allowed us to restrain the b and g torsion angles to 180 ± 30 and 54 ± 30 , respectively (24). The a, b and g torsion angles of residue U36 and z torsion angle of A35 were not restrained since they contain the phosphorus with a altered pattern in the 2D 1H-detected [1H,31P]HETCOR. The e torsion angles were restrained (to 230 ± 70 ) based on steric arguments (25). Hydrogen bond restraints in the stem were applied as NOE- distance restraints (residues C27!G31 and C39!G43). No restraints on c and no planarity restraints were used in the structure calculations. Structure determination All structure calculations were performed with X-PLOR-NIH V3.851 (26). The topallhdg.dna and parallhdg.dna ﬁles were adapted to include the modiﬁed residue using parameters obtained from ab initio calculations (27). In the topology ﬁle, a new t6A residue was introduced. The t6A residue was subsequently patched into the oligonucleotide sequence in a way comparable to the treatment of RNA and DNA in the standard X-PLOR program. The torsion angle molecular dynamics protocol used was largely identical to that proposed for a DNA duplex (28). A set of 100 structures was generated by torsion angle molecular dynamics, starting from an extended strand and using NMR derived restraints. After the torsion angle molecular dynamics round, the 39 structures had converged to very similar structures with similar total energies (189–381 kcal mol 1) and no violations of the NOE and dihedral restraints Twenty lowest energy structures were used for further reﬁnement during the ‘gentle molecular dynamics’ round. The ﬁnal reﬁnement started with a 20 ps constant- temperature molecular dynamics simulation at 300 K (20 000 steps of 0.001 ps) and was followed by a 200-step conjugate gradient energy minimization of the average structure of the last 10 ps of the 20 ps simulation. An analysis of the obtained 3D-structure with the computer programs X3DNA (29), was used to measure torsion angles and helix parameters. Finally, some visual representations of the molecule were obtained with Bobscript 2.4 (30). RESULTS Establishing optimal solution conditions Sharp resonances were observed at pH 7.4 and 5.5, indicative that the structure is stable in a single predominant conforma- tion in both conditions. Since pH 7.4 is closer to physiological conditions, we preferred to perform the structure determina- tion at this pH. Protonation of A38, which enhances the possibility of stable C32–A38+ base pair formation, was conﬁrmed by the charac- teristic shift of the A38 C2 resonance to (C2:154 p.p.m.! 148 p.p.m., H2:8.23 p.p.m.!8.33 p.p.m.) when lowering pH to 5.5 (31). However, there is no evidence that A38 is protonated at the physiological pH of 7.4. Assigment of resonances The ASL of S.pombe met-tRNAi was investigated by NMR using the chemically synthesized 17mer shown in Scheme 1. All aromatic, anomeric, H20 and H30 resonances could be assigned in the non-labeled sample starting from a standard anomeric to aromatic proton walk (32). Non-exchangeable protons of residues in the stem region showed NMR spectral characteristics typical of an A-form