EXPLORING THE AMINOACYLATION FUNCTION OF TRANSFER-RNA BY MACROMOLECULAR ENGINEERING APPROACHES - INVOLVEMENT OF CONFORMATIONAL FEATURES IN THE CHARGING PROCESS OF YEAST TRANSFER RNAASP

This report presents the conceptual and methodological framework that presently underlies the experiments designed to decipher the structural features in tRNA important for its aminoacylation by aminoacyl-tRNA synthetases. It emphasizes the importance of conformational features in tRNA for an optimized aminoacylation. This is illustrated by selected examples on yeast tRNAAsp. Using the phage T7 transcriptional system, a series of tRNAAsp variants were created in which conformational elements were modified. It is shown that aspartyl-tRNA synthetase tolerates conformational variability in tRNAAsp at the level of the D-loop and variable region, of the tertiary Levitt base-pair 15-48 which can be inverted and in the T-arm in which residue 49 can be excised. However, changing the anticodon region completely abolishes the aspartylation capacity of the variants. Transplanting the phenylalanine identity elements into a different tRNAAsp variant presenting conformational characteristics of tRNAPhe converts this molecule into a phenylalanine acceptor but is less efficient than wild-type tRNAPhe. This engineered tRNA completely loses its aspartylation capacity, showing that some aspartic acid and phenylalanine identity determinants overlap. The fact that chimeric tRNAAsp molecules with altered anticodon regions lose their aspartylation capacity demonstrates that this region is part of the aspartic acid identity of tRNAAsp. © 1990.