The identity determinants required for the discrimination between tRNA Glu and tRNA Asp by glutamyl‐tRNA synthetase from Escherichia coli

We previously elucidated the major determinant set for Escherichia coli tRNA Glu identity (U34, U35, C36, A37, G1•C72, U2•A71, U11•A24, U13•G22••Α46, and Δ47) and showed that the set is sufficient to switch the identity of tRNA Gln to Glu [Sekine, S., Nureki, O., Sakamoto, K., Niimi, T., Tateno, M., Go, M., Kohno, T., Brisson, A., Lapointe, J. & Yokoyama, S. (1996) J. Mol. Biol. 256 , 685–700]. In the present study, we attempted to switch the identity of tRNA Asp , which has a sequence similar to that of tRNA Glu , and consequently possesses many nucleotide residues corresponding to the Glu identity determinants (U35, C36, A37, G1•C72, and U11•A24). A simple transplantation of the rest of the major determinants (U34, U2•A71, U13•G22••Α46, and Δ47) to the framework of tRNA Asp did not result in a sufficient switch of the tRNA Asp identity to Glu. To confer an optimal glutamate accepting activity to tRNA Asp , two other elements, C4•G69 in the middle of the acceptor stem and C12•G23••C9 in the augmented D helix, were required. Consistently, the two base pairs, C4•G69 and C12•G23, in tRNA Glu had been shown to exist in the interface with glutamyl‐tRNA synthetase (GluRS) by phosphate‐group footprinting. We also found the two elements in the framework of tRNA Gln , and determined that their contributions successfully changed the identity of tRNA Gln to Glu in the previous study. By the identity‐determinant set (C4•G69 and C12•G23••C9 in addition to U34, U35, C36, A37, G1•C72, U2•A71, U11•A24, U13•G22••Α46, and Δ47) the activity of GluRS was optimized and efficient discrimination from the noncognate tRNAs was achieved.