A Watson-Crick Base-Pair-Disrupting Methyl Group (m 1 A9) Is Sufficient for Cloverleaf Folding of Human Mitochondrial tRNA Lys ² Mark Helm, Richard Giege ´, and Catherine Florentz* Unite ´ Propre de Recherche 9002 du CNRS, Institut de Biologie Mole ´ culaire et Cellulaire, 15 rue Rene ´ Descartes, F-67084 Strasbourg Cedex, France ReceiVed May 10, 1999; ReVised Manuscript ReceiVed July 21, 1999 ABSTRACT: We have previously shown by chemical and enzymatic structure probing that, opposite to the native human mitochondrial tRNA Lys , the corresponding in vitro transcript does not fold into the expected tRNA-specific cloverleaf structure. This RNA folds into a bulged hairpin, including an extended amino acid acceptor stem, an extra large loop instead of the T-stem and loop, and an anticodon-like domain. Hence, one or several of the six modified nucleotides present in the native tRNA are required and responsible for its cloverleaf structure. Phylogenetic comparisons as well as structural analysis of variant transcripts had pointed to m 1 A9 as the most likely important modified nucleotide in the folding process. Here we describe the synthesis of a chimeric tRNA Lys with m 1 A9 as the sole modified base and its structural analysis by chemical and enzymatic probing. Comparison of this structure to that of the unmodified RNA, the fully modified native tRNA, and a variant designed to mimic the effect of m 1 A9 demonstrates that the chimeric RNA folds indeed into a cloverleaf structure that resembles that of the native tRNA. Thus, due to Watson-Crick base-pair disruption, a single methyl group is sufficient to induce the cloverleaf folding of this unusual tRNA. This is the first direct evidence of the role of a modified nucleotide in RNA folding. Animal mitochondrial transfer RNAs (mt-tRNAs) 1 are well-known for a number of particular structural features which differentiate them from the defined canonical tRNAs (tRNAs from eukaryotic cytosols, eubacteria, archaebacteria, plant mitochondria, and chloroplasts as reviewed in ref 1). The most dramatic differences concern mt-tRNAs from nematodes all of which lack a complete structural domain, either the D-stem and loop or the T-stem and loop (2, 3). Along the same line, all mammalian tRNA Ser(AGY) are missing the D-stem and loop, which is replaced by a short connecting strand between the amino acid acceptor stem and the anticodon domain (4, 5). This holds true for tRNA Cys of some reptiles (6). Whereas all other animal mitochondrial tRNAs present a cloverleaf secondary structure, these structures, however, deviate from canonical tRNAs by changes in the size of individual stems and/or loops (for example, in the T-loop which is highly conserved in canonical tRNAs) and by the lack of conserved or semiconserved residues known as essential in the establishment of three-dimensional interac- tions (e.g., 1, 7). Thus, for example, many mt-tRNAs have A8 instead of U8, which hinders tertiary interaction with A14, and they miss the G18G19 sequence in the D-loop and the conserved TΨC sequence in the T-loop. Despite these structural abnormalities, mitochondrial tRNAs adopt three- dimensional structures allowing proper function as supported by experimental structural probing in the case of tRNA Ser (8-11) and sequence analysis combined with computer-aided model building (12-14). Animal mitochondrial tRNAs are further surprising at the level of their posttranscriptional modifications, probably as a consequence of their sequence and structure abnormalities. They are not only poorly but also differently modified as compared to canonical tRNAs. Whereas about 17% of nucleotides within cytosolic eukaryotic tRNAs are modified and 11% within eubacterial tRNAs, only about 6% are found modified in animal mitochondrial tRNAs (15). Moreover, the diversity of the modifications is much more restricted (15, 16). This suggests that these modifications are likely of higher importance than in the other tRNAs as they are retained by evolution. Interestingly, the greater importance of modified bases for efficient aminoacylation has already been observed in some mt-tRNAs as opposed to their cytosolic counterparts (e.g., 17-19, our unpublished results). Indeed, in vitro transcribed tRNAs are much poorer substrates for the aminoacyl-tRNA synthetases than the native modified tRNAs (e.g., 9). A structural analysis of human mitochondrial tRNA Lys lead us to the discovery of another particular structural feature never reported so far for canonical tRNAs. It was observed that the unmodified human mitochondrial tRNA Lys , corre- sponding in sequence to the mitochondrial gene, does not adopt the cloverleaf structure of the fully modified tRNA. Hence, it can be assumed that modified bases are required to reach its appropriate secondary structure (20). This unexpected requirement is opposite to what is found in all canonical tRNAs for which in vitro transcribed versions, ² This investigation was supported by the Centre National de la Recherche Scientifique (CNRS), the Universite ´ Louis Pasteur (Stras- bourg), and the Association Franc ¸ aise contre les Myopathies (AFM). M.H. was supported by a Marie-Curie fellowship from the EC TMR program. * To whom correspondence should be addressed. Telephone: 33 3 88 41 70 59. Fax: 33 3 88 60 22 18. Email: florentz@ibmc.u-strasbg.fr. 1 Abbreviations: DEPC, diethyl pyrocarbonate; DMS, dimethyl sulfate; mt, mitochondrial; tRNA, transfer RNA. 13338 Biochemistry 1999, 38, 13338-13346 10.1021/bi991061g CCC: $18.00 © 1999 American Chemical Society Published on Web 09/11/1999