uridine riboses still interconvert between 20 endo and 30 endo conformations because of weak uridine stacking. The crystal 5 5 structure of human lys-tRNAUUU that has mcm s U34 and ms2t6A37 modiﬁcations in its anticodon loop, demonstrates that when anticodon residues are base paired their ribose conformation becomes 100% 30 endo within the context of an A-type RNA duplex (46). However, the presence of the typical ‘tRNA-like’ structure with three anticodon nucleotides positioned to bind the cognate codon to form an A-type double helical RNA complex with mRNA (Figure 5C) is not required for protein synthesis, 6 since presence of t A37 in ASL of lys-tRNAUUU enables to bind AAA in the A-site of the ribosome (12), a feature that is absent for the unmodiﬁed lys-tRNA stem–loop (47). Combination of modiﬁcations, t6A37 and mnm 5 s 2 U34, in ASL of lys-tRNAUUU enables binding to AAA and AAG codons in the ribosome (12). Such a wobbling does not occur with met-tRNAi since it only initiates translation at an AUG start- codon in vivo. Critically important in lys-tRNAUUU is that the base modiﬁcation negates the formation of an intraloop U33.A37 base pair that as is observed in the unmodiﬁed ASL. This therefore reduces the energetic cost for base exposure during codon–anticodon binding. In the structure of the E.coli tRNAi residues A37 and U33 are coplanar but no base pairing occurs between them according to the measured dis- tances. The presence of a tricylic t6A in our structure slightly displaces U33 further away from its position in the unmodiﬁed loop, negating the possibility of an U33.t6A37 base pair in the loop. CONCLUSION The anticodon loop is a region with high conformational diversity dependence on sequence, the presence of modiﬁed nucleosides and its environment. The t6A modiﬁcation of tRNA in the ASL, 30 adjacent to the anticodon, is known to facilitate codon recognition and negate translational frame 6 shifting. The presence of t A37 in the ASL of met-tRNAi reduces its ﬂexibility and causes rearrangements of the loop structure that adopts a type of U-turn that was not observed previously. Structural changes caused by the presence of the 6 t A modiﬁcation do not result in the typical ‘tRNA-like’ struc- ture with three anticodon nucleotides positioned to bind the cognate codon to form an A-type double helical RNA com- plex with mRNA. However, previous studies on the ASL of tRNAUUU demonstrated that such a highly pre-organized structure of the anticodon loop is not required for codon- binding, but induces wobbling at the third codon position. Such a wobbling is not desired for met-tRNAi, since it would cause inaccuracy in the initiation of protein synthesis. While the unmodiﬁed anticodon loop can be considered as a point of structural dynamics in the prokaryotic met-tRNAi 6 molecule, the threonyl side chain of t A37 in eukaryotic met-tRNAi creates a tricyclic nature of t6A that enhances stacking interactions of the loop and stem region. Additional hydrogen bonds of this threonyl side chain with different nucleosides in the loop also increase loop stability. Because the threonyl moiety must accommodate within the loop, U33 is slightly shifted and U36 is completely bulged out. The latter Nucleic Acids Research, 2006, Vol. 34, No. 10 2885 enables efﬁcient A35 to t6A37 stacking following a U-turn. Here, we demonstrated that a unique U-turn motif in a hairpin loop can be obtained by introducing one modiﬁed nucleoside (t6A). When evaluating the structure of the ASL of tRNAi of S.pombe, the modiﬁed t6A37 might help in presenting decod- ing bases outside the loop structure which is not the case for the unmodiﬁed loop and cannot be concluded from the X-ray structure. Whether the bulged U36 has a functional role in initiating the recognition process leading to eukaryote translation remains a subject for further studies. ACKNOWLEDGEMENTS The authors thank Brian Sproat for the synthesis of the oligo- mers and Ingrid Luyten for initial experiments. E.L. and K.N. are Research fellows of the Fund for Scientific Research— Flanders (Belgium). The authors thank the European NMR Large Scale Facility in Frankfurt for access to the 800 MHz spectrometer. The refined coordinates of the twenty structures closest to the average are deposited in the Brookhaven Protein Data Bank (2G1G.pdb) together with the measured chemical shifts and experimental restraints. 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