Nucleic Acids Research, 2006, Vol. 34, No. 10 2883 A B Figure 4. 1D 1H (A) and 31P (B) spectra at different Mg2+ concentrations. H5 and H10 signals close to the water are influenced by the applied water-suppression (watergate). Signals that are significant for the loop structure are indicated. hexaloop in ribosomal RNA are reported in literature. In the hairpin structure determined by Huang et al. (40) the consec- utive residues UAA adopt a U-turn stacked on a non-canonical GA base pair while the last uridine in the loop is bulged out. However, Fountain et al. (41) describes the structure of this loop with two non-canonical base pairs (GA and UU) and two unpaired adenines. It is possible that the increased concentra- tion of salts used by these authors induced the conformational change in their loop: the structure of Huang et al. (40) was determined from a 1.5 mM hairpin sample at pH 6 (5 mM sodium phosphate buffer) containing 30 mM NaCl and 0.04 mM EDTA while Fountain et al. (41) used a 1.8 mM oligomer sample at pH 7 (10 mM sodium phosphate buffer) with 100 mM NaCl and 0.5 mM EDTA. A slightly modiﬁcation of a ‘classical’ U-turn occurs start- ing from residue U33 (Scheme 2). The U-turn is an RNA structural motif that was ﬁrst identiﬁed in the crystal structure of the yeast Phe-tRNA anticodon loop (42) and is now recog- nized as a basic architectural unit of many RNA molecules. This RNA motif reverses the direction of the phosphate backbone, frequently 30 to a uridine nucleotide. The turn is achieved by adoption of the nonstandard trans conformation of the a torsion angle 30 to U33. It is stabilized by stacking 6 interactions of U33 with C32 and between A35 and t A37. The hydrogen bond between U33:H3 and the phosphate group of U36, known to stabilize a U-turn (25), are not present in the studied ASL due to the altered position of U36. However atoms U33:O20 and A35:N7 are within hydrogen bonding distance (2.68 s) as expected in a U-turn (25). As in a ‘classical’ U-turn, the Watson–Crick faces of both nucleo- bases following the turning phosphate are exposed towards the solvent and prone to interact with cognate bases of mRNA. Table 2. Atom pairs in the loop of the calculated structures that are within hydrogen bonding distance H-donor t6A37:OH14 C34:N4 U33:O20 C32:N4 H-acceptor C32:O2 t6A37:O13a A35:N7 A38:N1 distance 1.35 (0.02) 2.47 (0.15) 2.89 (0.07) 2.64 (0.04) Distances and their RMSD in the 20 selected structures are listed in A˚ . aO13A or O13B, whichever is turned towards C34:N4. Comparison with relevant tRNA structures Based on the RMSD difference of modiﬁed and unmodiﬁed structures, introduction of a t6A modiﬁcation restricts the con- formational space of the anticodon loop (unmodiﬁed: 0.3– 0.9 s, modiﬁed: 0.09–0.23 s). The loop structure we deter- mined is not only better deﬁned, but also differs signiﬁcantly from its unmodiﬁed relative depicted in the left panel of Figure 1 (13). The ﬁve Watson–Crick base pairs and a non- canonical C32.A38 base pair in the stem of both structures are in the standard A-form of helical RNA. The t6A37 adjacent to the anticodon adopts the form of a tricyclic nucleoside with an intra-residue hydrogen-bond and alters base stacking on the 30- side of the anticodon loop. U36 of the anticodon is signiﬁc- antly displaced by the presence of t6A and allows A35 to stack 6 on t A37. Both modiﬁed and unmodiﬁed loops contain a U-turn motif, formed by residues U33, C34 and A35. However the residue stacking on the 30 side of the U-turn differs: in the unmodiﬁed loop this stacking occurs with U36 while bulging out of U36 by the t6A modiﬁcation induces stacking of A35 on 6 t A37, which in turn stacks on the adjacent stem. Apparently,