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Volume 272, Number 36, Issue of September 5, 1997 pp. 22809-22816
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Human Lysyl-tRNA Synthetase Accepts Nucleotide 73 Variants and Rescues Escherichia coli Double-defective Mutant

(Received for publication, February 20, 1997, and in revised form, June 7, 1997)

Kiyotaka Shiba ^§ , Timothy Stello , Hiromi Motegi ^§ , Tetsuo Noda ^§ , Karin Musier-Forsyth and Paul Schimmel ^**

From the ^§ Department of Cell Biology, Cancer Institute, Japanese Foundation for Cancer Research, Kami-Ikebukuro, Toshima-ku, Tokyo 170, Japan,  Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Corporation, the  Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, and the ^** Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

The nucleotide 73 (N⁷³) "discriminator" base in the acceptor stem is a key element for efficient and specific aminoacylation of tRNAs and of microhelix substrates derived from tRNA acceptor stems. This nucleotide was possibly one of the first to be used for differentiating among groups of early RNA substrates by tRNA synthetases. In contrast to many other synthetases, we report here that the class II human lysyl-tRNA synthetase is relatively insensitive to the nature of N⁷³. We cloned, sequenced, and expressed the enzyme, which is a close homologue of the class II yeast aspartyl-tRNA synthetase whose co-crystal structure (with tRNA^Asp) is known. The latter enzyme has a strong requirement for G⁷³, which interacts with 4 of the 14 residues within the "motif 2" loop of the enzyme. Even though eukaryotic lysine tRNAs also encode G⁷³, the motif 2 loop sequence of lysyl-tRNA synthetase differs at multiple positions from that of the aspartate enzyme. Indeed, the recombinant human lysine enzyme shows little preference for G, and even charges human tRNA transcripts encoding the A⁷³ found in E. coli lysine tRNAs. Moreover, while the lysine enzyme is the only one in E. coli to be encoded by two separate genes, a double mutant that disables both genes is complemented by a cDNA expressing the human protein. Thus, the sequence of the loop of motif 2 of human lysyl-tRNA synthetase specifies a structural variation that accommodates nucleotide degeneracy at position 73. This sequence might be used as a starting point for obtaining highly specific interactions with any given N⁷³ by simple amino acid replacements.

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