Twenty-first aminoacyl-tRNA synthetase-suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins ineukaryotes and in eubacteria

Two critical requirements for developing methods for the site-specific inco rporation of amino acid analogues into proteins in vivo are (i) a suppresso r tRNA that is not aminoacylated by any of the endogenous aminoacyl-tRNA sy nthetases (aaRSs) and (ii) an aminoacyl-tRNA synthetase that aminoacylates the suppressor tRNA but no other tRNA in the cell. Here we describe two suc h aaRS-suppressor tRNA pairs, one for use in the yeast Saccharomyces cerevi siae and another for use in Escherichia coli. The "21st synthetase-tRNA pai rs" include E. coli glutaminyl-tRNA synthetase (GlnRS) along with an amber suppressor derived from human initiator tRNA, for use in yeast, and mutants of the yeast tyrosyl-tRNA synthetase (TyrRS) along with an amber suppresso r derived from E. coli initiator tRNA, for use in E. coli. The suppressor t RNAs are aminoacylated in vivo only in the presence of the heterologous aaR Ss, and the aminoacylated tRNAs function efficiently in suppression of ambe r codons. Plasmids carrying the E. coli GlnRS gene can be stably maintained in yeast. However, plasmids carrying the yeast TyrRS gene could not be sta bly maintained in E. coli. This lack of stability is most likely due to the fact that the wild-type yeast TyrRS misaminoacylates the E. coli proline t RNA. By using error-prone PCR, we have isolated and characterized three mut ants of yeast TyrRS, which can he stably expressed in E. coli. These mutant s still aminoacylates the suppressor tRNA essentially quantitatively in viv o but show increased discrimination in vitro for the suppressor tRNA over t he E. coli proline tRNA by factors of 2.2- to 6.8-fold.