The identity determinants required for the discrimination between tRNAGlu and tRNAAsp by glutamyl-tRNA synthetase from Escherichia coli.
We previously elucidated the major determinant set for Escherichia coli tRNAGlu identity (U34, U35, C36, A37, G1*C72, U2*A71, U11*A24, U13*G22**Alpha46, and Delta47) and showed that the set is sufficient to switch the identity of tRNAGln 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 tRNAAsp, which has a sequence similar to that of tRNAGlu, 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**Alpha46, and Delta47) to the framework of tRNAAsp did not result in a sufficient switch of the tRNAAsp identity to Glu. To confer an optimal glutamate accepting activity to tRNAAsp, 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 tRNAGlu 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 tRNAGln, and determined that their contributions successfully changed the identity of tRNAGln 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**Alpha46, and Delta47) the activity of GluRS was optimized and efficient discrimination from the noncognate tRNAs was achieved.
Sekine
S
S
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Japan.
Nureki
O
O
Tateno
M
M
Yokoyama
S
S
D000215:Acylation; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D012349:RNA, Transfer, Asp / Q000737:chemistry* / Q000235:genetics; D012352:RNA, Transfer, Glu / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity
D012349:RNA, Transfer, Asp; D012352:RNA, Transfer, Glu; D000604:Amino Acyl-tRNA Synthetases