THE PRESENCE OF MODIFIED NUCLEOTIDES IS REQUIRED FOR CLOVERLEAF FOLDING OF A HUMAN MITOCHONDRIAL TRANSFER-RNA

Direct sequencing of human mitochondrial tRNA(Lys) shows the absence o f editing and the occurrence of six modified nucleotides (m(1)A9, m(2) G10, Psi 27, Psi 28 and hypermodified nucleotides at positions U34 and A37), This tRNA folds into the expected cloverleaf, as confirmed by s tructural probing with nucleases, The solution structure of the corres ponding in vitro transcript unexpectedly does not fold into a cloverle af but into an extended bulged hairpin. This non-canonical fold, estab lished according to the reactivity to a large set of chemical and enzy matic probes, includes a 10 bp aminoacyl acceptor stem (the canonical 7 bp and 3 new pairs between residues 3-10 and 65-63), a 13 nt large l oop and an anticodon-like domain. It is concluded that modified nucleo tides have a predominant role in canonical folding of human mitochondr ial tRNA(Lys). Phylogenetic comparisons as well as structural probing of selected in vitro transcribed variants argue in favor of a major co ntribution of m(1)A9 in this process.