Transfer RNA-mediated suppression of stop codons in protoplasts and transgenic plants

Abstract

We have developed a simple, rapid and sensitive assay for tRNA gene expression in plant cells. A plant tRNA(Leu) gene was site-specifically mutated to encode each of the three anticodon sequences (CUA, UUA and UCA) that recognize, respectively, the amber, ochre and opal stop codons. The suppression activity of these genes was detected by their ability to restore transient beta-glucuronidase (GUS) expression in tobacco protoplasts electroporated with GUS genes containing premature stop codons. Protoplasts co-electroporated with the amber suppressor tRNA gene and a GUS gene containing a premature amber stop codon showed up to 20-25% of the activity found in protoplasts transfected with the functional control GUS gene. Ochre and opal suppressors presented maximum efficiencies of less than 1%. This system could be adapted to examine transcription, processing or aminoacylation of tRNAs in plant cells. In addition, phenotypically normal, fertile tobacco plants expressing a stably incorporated amber suppressor tRNA gene have been obtained. This suppressor tRNA can be used to transactivate a target gene containing a premature amber stop codon by a factor of at least several hundred-fold.