2882 Nucleic Acids Research, 2006, Vol. 34, No. 10 The sugar moieties of these residues were therefore not restrained during the structure calculations. Six negative distance constraints (33) with a lower bound of 4.0 s, derived from absent crosspeaks in the measured NOESY spectra, were used during the ﬁrst round of structure calculation. Hydrogen bonds in base pairs were treated as NOE distance restraints between the acceptor and donor heavy atoms. The structure of the RNA hairpin was solved from 280 distance and 117 torsion angle restraints. Starting from an extended strand, a set of 100 structures was generated by torsion angle dynamics. Out of these 39 structures converged that had no NOE restraint violations >0.5 s and no torsion angle restraint violations >5 (Table 1). These 39 accepted structures were subjected to reﬁnement in a ﬁnal round of energy minimization. The twenty lowest energy structures were selected for structural analysis (Table 1). Figure 3 shows the superposition of the 20 structures illustrating well-deﬁned structures of the loop and stem region. Binding of Mg2+ was monitored by NMR to detect speciﬁc binding sites in the loop region of the duplex. However, addition of Mg2+ did not induce signiﬁcant chemical shift changes in 31P or 1H signals of the loop (Figure 4). Only slight chemical shift changes of loop signals occurred in the 1H spectrum at increasing Mg2+ concentration from 0 to 1 mM. Notably, 31P signals characteristic for the U-turn (U33:P) and the bulged-out U36 in the anticodon loop (A35 and U36:P), did not shift signiﬁcantly when Mg2+ was added to the sample. These results indicate that there is no major conformational change within the anticodon loop that could be caused by speciﬁc Mg2+ binding. Absence of tight Mg2+ binding in the studied ASL is in agreement with the very weak Mg2+ induced cleavage of this loop observed for Yeast met-tRNAi that was attributed to low afﬁnity of metal ions or a less favorable structure of this loop for efﬁcient cleavage (36). DISCUSSION Structure of the ASL of S.pombe As expected for double stranded RNA, the stem-region adopts a typical A-form structure. There is no direct evidence that Table 1. Refinement statistics for the final set of 20 structures and overview of the NMR constraints used during structure calculation Total energy (kcal mol 1) NOE violations (>0.5 s) NOE constraints inter-residue intra-residue base pairing non-noe Dihedral violations (>5 ) Torsion angle constraints RMSD from distance restraints (A˚ ) RMSD from dihedral restraints ( ) RMSD in A˚ omitting the closing base pair (U27–A43) 418.5 ± 3.6 0 274 (280) 108 151 15 6 1 ± 1 117 0.059 ± 0.001 0.837 ± 0.038 0.09–00.24 Non-noe constraints were used during the torsion angle dynamics but omitted in the refinement stage Reported RMSD is the average of all heavy atoms in a refined structure relative to the average of the 20 selected structures (0.09 s ¼ structure closest to the average of refined structures). A38 is protonated (at pH 7.4) and involved in an A38+.C32 pair. However, much indirect evidence seems to point to it and resulted in a structure that contains a A38:C32 base pair. The NMR data indicate that the stem is extended beyond the G31·C39 pair and that A-form stacking continues on the 50 side and as well as on 3’ side. Although no assumptions or explicit hydrogen bond restraints were introduced to force the A38– C32 mismatch (Figure 2, left), in the 39 reﬁned structures A38:N1 and C32:N4 are within hydrogen bonding distance (<3.0 s) to form an A:C N1-amino base pair (37) (Table 2). The loop portion was very well-deﬁned by the NMR 6 restraints of the threonine moiety in t A37 (Figure 3) and 11 NOE interactions of base protons in A35. Structure calcu- lations therefore resulted in a highly deﬁned structure that shows t6A37:O13A and t 6 A37:OH14 within hydrogen bond- ing distance of C34:N4 and C32:O2, respectively (<3.0 s) (Figure 2, right). As in GNRA tetraloops, whose NMR struc- tures show low pairwise RMSD (33), a network of heterogen- eous hydrogen bonds and extensive base stacking in the loop contribute to the highly deﬁned loop-structure. The residue U36 is completely bulged out while A35 is 6 stacking on t A37. This rather unexpected structural feature is supported by the observed NOE-contacts and the upﬁeld chemical shift of U36:H5 that is in agreement with absent stacking interactions of a bulged out pyrimidine while the A35:H8 signal is in the region typical for an A-type helix (35). As depicted in Figure 2 (left), the tricyclic t6A37 nuc- leobase (green) is stacked between A35 (yellow) and A38 (grey) while U36 is completely bulged out. The helical twist of A35 relative to t6A37 in the structure closest to the average of all reﬁned structures is 33.79 (Curves: Lavery and Sklenar) (38), a value that is comparable to the 31.4 s found in A-type RNA (25). The bulged out U36 residue exposes its nucleobase into the solvent, making it available for possible tertiary interactions. That U36 would have a natural tendency to bulge out was suggested in literature reports since it is an unpaired uridine (39) and it is the n + 3 residue in a U-turn (below). Conﬂicting structures of the conserved GUAAUA Figure 3. Stereo overlay of the 20 lowest energy structures fitted to all residues except those in the closing base pair (U27–A43).