HUMAN LYSYL-TRANSFER-RNA SYNTHETASE ACCEPTS NUCLEOTIDE-73 VARIANTS AND RESCUES ESCHERICHIA-COLI DOUBLE-DEFECTIVE MUTANT

The nucleotide 73 (N-73) ''discriminator'' base in the acceptor stem i s a key element for efficient and specific aminoacylation of tRNAs and of microhelix substrates derived from tRNA acceptor stems, This nucle otide was possibly one of the first to be used for differentiating amo ng groups of early RNA substrates by tRNA synthetases, In contrast to many other synthetases, we report here that the class II human lysyl-t RNA synthetase is relatively insensitive to the nature of N-73. We clo ned, sequenced, and expressed the enzyme, which is a close homologue o f the class II yeast aspartyl-tRNA synthetase whose co-crystal structu re (with tRNA(Asp)) is known. The latter enzyme has a strong requireme nt for G(73), which interacts with 4 of the 14 residues within the ''m otif 2'' loop of the enzyme. Even though eukaryotic lysine tRNAs also encode G(73), the motif 2 loop sequence of lysyl-tRNA synthetase diffe rs at multiple positions from that of the aspartate enzyme, Indeed, th e recombinant human lysine enzyme shows little preference fair G, and even charges human tRNA transcripts encoding the A(73) found in E. col i 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 disab les both genes is complemented by a cDNA expressing the human protein. Thus, the sequence of the loop of motif 2 of human lysyl-tRNA synthet ase specifies a structural variation that accommodates nucleotide dege neracy at position 73, This sequence might be used as a starting point for obtaining highly specific interactions with any given N-73 by Sim ple amino acid replacements.