2878–2886 Nucleic Acids Research, 2006, Vol. 34, No. 10 doi:10.1093/nar/gkl081 The naturally occurring N6-threonyl adenine in anticodon loop of Schizosaccharomyces pombe tRNAi causes formation of a unique U-turn motif Eveline Lescrinier, Koen Nauwelaerts, Katia Zanier1, Koen Poesen 1 , Michael Sattler1 and Piet Herdewijn* Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, BioMacs, K.U.Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgiumand and 1EMBL, Structural & Computational Biology and Gene Expression, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Received December 23, 2005; Revised January 23, 2006; Accepted March 6, 2006 ABSTRACT Modified nucleosides play an important role in struc- ture and function of tRNA. We have determined the solution structure of the anticodon stem–loop (ASL) of initiator tRNA of Schizosaccharomyces pombe. The incorporation of N6-threonylcarbamoyladen- osine at the position 30 to the anticodon triplet (t6A37) results in the formation of a U-turn motif and enhances stacking interactions within the loop and stem regions (i.e. between A35 and t6A37) by bulging out U36. This conformation was not observed in a crystal structure of tRNAi including the same modification in its anticodon loop, nor in the solution 6 structure of the unmodified ASL. A t A modification also occurs in the well studied anti-stem–loop of lys- tRNAUUU. A comparison of this stem–loop with our structure demonstrates different effects of the modification depending on the loop sequence. INTRODUCTION A special methionine tRNA is used for the initiation of protein synthesis in all organisms. Two classes of methionine tRNA are found universally: the initiator tRNA (tRNAi) that is used exclusively for translation initiation and elongator tRNA (met-tRNAe) used for insertion of methionine into internal peptidic linkages (1,2). Distinct mechanisms of translation initiation are used by prokaryotes and eukaryotes. In prokaryotes, the small ribosomal subunit (30S) forms an initiation complex with formylmethionine tRNA (fmet-tRNAi) and a GTP binding protein called IF2 at certain AUG or AUG-cognate codons. In addition to IF2, also initiation factors IF3 and IF1 are required for initiation (3). Both of these factors stabilize the binding of fmet-tRNAi·IF2 to the 30S subunit (4). IF3 contributes to the ﬁdelity of initiation complexes that might form transiently at weak sites such as non-AUG codons (5). In cytoplasmic protein synthesis of eukaryotes, the small ribosomal subunit (40S) normally enters at the 50 end of the mRNA and not at the AUG start-codon. The 40S subunit, carrying methionyl tRNA (met-tRNAi), eIF2, GTP and other factors, then migrates through the 50-untranslated region (50-UTR) until it encounters the ﬁrst AUG codon, which is recognized by base pairing with the anticodon met-tRNAi. Selection of the start-codon is ﬁxed when a large ribosomal subunit (60S) joins the paused 40S subunit (3). In contrast with prokaryotic ribosomes, which often can also initiate at GUG or UUG, the eukaryote initiation mechanism does not allow an alternative codon to substitute for AUG in vivo. In yeast, the dominant role of every position of the anticodon in deﬁning the initiator codon was demonstrated by changing the anticodon in met-tRNAi to 30-UCC-50 and showing that translation initiated exclusively at the ﬁrst AGG codon in a test transcript (6). A strong codon–anticodon interaction is therefore prerequisite for correct translation initiation in the eukaryotic cytoplasm. In all cytoplasmatic met-tRNAi of eukaryotes and plants a threonylcarbamoyl modiﬁcation occurs at the 30 site of the anticodon (t6A37) whereas this nucleoside modiﬁcation does not occur in fmet-tRNAi of prokaryotes, chloroplasts and archaeabacteria. It has been proposed that one of the 6 roles of t A37 is to prevent mispairing between the ﬁrst base of the codon and the third base of the anticodon (7). *To whom correspondence should be addressed. Tel: +32 0 16 337387; Fax: +32 0 16 337340; Email: piet.herdewijn@rega.kuleuvan.ac.be The Author 2006. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org