2884 Nucleic Acids Research, 2006, Vol. 34, No. 10 the modiﬁcation structures the loop by extra hydrogen bonds of the threonyl side chain with several loop-residues and increased stacking on the stem at its 30 side. All three nucleo- bases of the anticodon in the modiﬁed loop have their Watson- Crick binding faces exposed to the solvent. In the unmodiﬁed hairpin, the loop also presents three anticodon bases for codon- binding, although their positions are not as well-deﬁned and stacking between U36 and A37 is absent. The ASL in the crystal structure of S.cerevisiae met-tRNAi (1YFG, right panel of Figure 1) is signiﬁcantly different from the solution structure we describe here: t6A37 is bulged out, a U-turn is formed by U33-C34-A35 and all three anticodon nucleobases (C34,A35,U36) are stacked on top op of A38, extending the 50 end of the stem. However, according to the authors, the anticodon loop in their crystal structure is not well enough deﬁned to discuss conﬁdently the implications of its structure in initiation of translation. Moreover, the anticodon in this structure is involved in lattice contacts, which may have imposed a stereochemistry that may not exist in solution (14). As in the structure we determined, the t6A modiﬁcation in the ASL of lys-tRNAUUU (1FEQ, Figure 5B) is t6A stacking on 50 side of the stem and has a tricyclic nature by an intraresidue hydrogen bond of the threonyl side chain with N7 of adenine (11). Durant et al. (43) also studied this loop-modiﬁcation and report that t6A decreases anticodon stacking in the loop by promoting U36 bulging. However, also in their structure U36 is not completely bulged out as what we observe in the modi- ﬁed initiator tRNA-loop. The A35-t6A37 stacking in our struc- ture clearly contributes to stabilization of a structure with bulged out U36. In the unmodiﬁed ASL of in lys-tRNAUUU (2BZ1, Figure 5A), the loop is rather dynamic with only weak stacking between U35–U36 and no NOEs that would indicate base stacking between U34 and U35 (44). The poor stacking was attributed to the loop sequence composed of consecutive uridines that are known to have weak stacking interactions (25). In this unmodiﬁed loop, non-speciﬁc Mg2+ (15 mM) apparently promotes U36-A37-A38 stacking. A high resolu- tion NMR structure is also available for the hyper-modiﬁed ASL of E.coli lys-tRNAUUU5 w2ith t6A and 5-methylamino- methyl-,2-thiouridine (mnm s U34) in the loop section (Figure 5D) (45). The 31P spectrum of this loop has 33p34 shifted signiﬁcantly downﬁeld, consistent with U-turn geo- metry where nucleobases of the 30 side of the loop starting at position 34 are stacked in an A-form geometry with the double stranded stem beginning at residue 39. Apparently, the presence of only t6A in 1FEQ does not induce this U-turn and U34 in lys-tRNAUUU is displaced in the loop, away from other anticodon uridines and is not exposing its Watson–Crick face to the solvent. The hypermodiﬁcation induces formation of a nearly canonical tRNA anticodon loop; however, the loop Scheme 2. Representation of a ‘classical’ U-turn (left) (25) and the U-turn in the ASL of S.pombe met-tRNAi (right) on left and right panels, respectively. R represents a purine and N a purine or pyrimidine nucleobase. Stabilizing hydrogen bonding interactions of the U-imino with 50 phosphate following the UNR sequence and of 20 hydroxyl of U with R:N7 are indicated in red dashed lines. Atoms U33:O2’ and A35:N7 that are within hydrogen bonding distance are also connected by a red dashed line. Figure 5. (A) 2BZ1: the unmodified ASL of Lys-tRNAUUU) (43), (B) 1FEQ: hairpin structure with the same sequence including t6A modification at position 37 (11), (C) 1· MQ: the same ASL as in 1FEQ and cognate mRNA (red) as they occur in the A-site of the crystal structure bound to a 30S ribosomal subunit (12) and (D) 1FL8 : hypermodified ASL with same nucleoside sequence including mnm 5 s 2 U34, t6A37 and Y39 (44). The right structure is determined by christallography, others are determined by NMR spectroscopy. Ribbons are drawn through P atoms and nucleobases of the UUU-anticodon are depicted in yellow.