This idea was essentially based on the observation that Escherichia coli fmet-tRNAi recognizes not only the AUG initiator codon ( 90%), but also GUG ( 8%) and UUG ( 1%), while yeast fmet-tRNAi with t6A37 modiﬁcation exclusively recognizes AUG in vivo (8,9). In thermodynamic and kinetic studies on yeast arg-tRNA it was shown that a 6 t A37 modiﬁcation has a stabilizing effect on U-A and U-G base pairs adjacent to the 50-side of the modiﬁed nucleoside (10) in vitro. The solution structure showed that t6A37 pre- vents the formation of a U33–A37 base pair that would force the anticodon loop towards an unfavorable conformation which would increase the entropic cost of tRNA binding (11). The crystal structure of modiﬁed lys-tRNAUUU anticodon stem–loops (ASL) bound to a 30S ribosomal subunit revealed a second way in which t6A37 is favorable for codon–anticodon interaction: (12) the extended ring structure of t6A37 enables cross-strand base-stacking of this modiﬁed nucleoside in the anticodon loop with A38 of tRNA and with A1, the base in the ﬁrst position of the codon. This stacking arrange- ment helps to explain the observed increase in stability of the codon–anticodon interaction of the modiﬁed ASL versus that of the unmodiﬁed case. RNA base pairing between the initiation codon and anti- codon loop of initiator tRNA is essential but not sufﬁcient for the selection of the ‘correct’ mRNA translational start-site by ribosomes. However, an anticodon:codon interaction between met-tRNAi and the ﬁrst AUG, has a function in directing the ribosome to the eukaryotic initiator region as part of the scan- ning ribosome. The structure of the ASL of met-tRNAi is therefore of biological interest. The solution structure of the ASL of E.coli without modiﬁed nucleosides, was determined by Schweisguth et al. (13) A crystal structure of the entire eukaryotic tRNAi of Saccharomyces cerevisiae is available at the pdb databank (1YFG) but fails to reveal structural details of the ASL with its t6A37 (14). We studied the ASL of Schizosaccharomyces Pombe (15), a unicellular eukaryote that is used as a model organism in molecular and cell biology, to determine the inﬂuence of the t6A modiﬁcation on the structure of the isolated ASL in eukaryotic tRNAi. t6A is Nucleic Acids Research, 2006, Vol. 34, No. 10 2879 the only modiﬁed nucleoside present in the ASL of met-tRNAi in S.pombe (Scheme 1). MATERIALS AND METHODS NMR sample preparation NMR sample conditions were 10 mM potassium phosphate (pH 7.4) with RNA concentrations of 0.6 mM. Samples were annealed prior to NMR experiments by brieﬂy heating at 80 C and snap cooling on ice to promote hairpin formation. For spectra in D2O, samples were lyophilized and dissolved in 100% D2O. NMR spectroscopy Natural abundance [1H,13C]HSQC and NOESY spectra in D2O were recorded on a Bruker DRX800 equipped with a 5 mm TXI HCN Z gradient cryoprobe (Centre for Biomolecu- lar Magnetic Resonance, BMRZ, University, Frankfurt). Phosphorus spectra were obtained on a Bruker DRX600 spectrometer equipped with a broadband TBI probe. Other spectra are measured with our ‘in house’ Varian Unity 500 spectrometer using a 3 mm HCPzgrd probe. Unless stated differently, spectra were recorded at 22 C. Spectra were processed using the FELIX 97.00 software package (Bio- sym Technologies, San Diego, VA (Accelrys )) running on a Silicon Graphics O2 R10000 workstation (IRIX version 6.3). The 1D spectrum in H2O was recorded using a jump-return pulse as the observation pulse (16). The 2D NOESY in H2O (mixing time ¼ 200 ms; at 5 C) was recorded using the WATERGATE water-suppression (17) with a sweep width of 10 000 Hz in both dimensions, 64 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 2D DQF-COSY (18), TOCSY (19) and NOESY (20) spectra in D2O were recorded with a sweep width of 4200 Hz in both dimensions. The DQF-COSY spectrum consisted of Figure 1. NMR structure of the unmodified ASL of E.coli tRNAi (residues 3 to 19) (13) (A), hairpin structures with the same loop-sequence including t6A modification at position 37 (green) (S.pombe tRNAi, structure closest to the average of refined NMR ensemble) (B) and X-ray crystallography (S.cerevisiae tRNAi residues 27 to 43) (14) (C). Ribbons are drawn through P atoms and nucleobases of the anticodon are depicted in yellow.