In vivo aminoacylation of human and Xenopus suppressor tRNAs constructed by site-specific mutagenesis.

Abstract

Amber suppressor tRNA genes were constructed by site-specific mutagenesis of the anticodons of human lysine-inserting tRNA (tRNALys) and glutamine-inserting tRNA (tRNAGln) genes, and Xenopus laevis tyrosine-inserting tRNA (tRNATyr) gene. As previous in vitro studies in prokaryotes have shown that substitution of nucleotides in the anticodon region can profoundly affect tRNA aminoacylation, it is important to determine whether the mutation affects aminoacylation of these eukaryotic tRNAs. We present a method for quantitating the tRNA aminoacylation in vivo in mammalian cells, and we have determined that the suppressor tRNATyr is fully aminoacylated and suppressor tRNALys and tRNAGln are aminoacylated 40-50% and 80%, respectively. This in vivo method of estimating aminoacylation may be applied to other mutations in the tRNA genes.