Regulated expression of plant tRNA genes by the prokaryotic tet and lac repressors

The prokaryotic tet operator (tetO) sequence was inserted at positions upstream and downstream of sequences encoding the Arabidopsis thaliana tRNAAUCLys or tRNAAUCTrp suppressor tRNAs, and tRNA expression in carrot protoplasts was measured by translational suppression of a nonsense codon in a luciferase reporter gene. Regulation of tRNA expression by the tetracycline repressor (tetR) occurred from genes with the tetO inserted at position −1 (for the tRNAAUCTrp gene), or at positions −2, −6 and −10 (for the tRNAAUCLys gene), and repression reached 90%. The inducer tetracycline (Tc) restored tRNA expression. Similarly, carrot protoplasts transfected with human tRNAAUCSer genes containing the lac operator (lacO) in their 5′-flanking sequence with or without the lac repressor (lacI) gene, conditionally expressed tRNAs which suppressed the luciferase reporter. Up to 30-fold repression occured by the lactose repressor when lacO was located at position −1 of the tRNAAUCSer coding sequence. In the presence of the inducer isopropyl-β-thiogalactoside (IPTG), repression was relieved. These results demonstrate that sequences flanking tRNA genes can strongly influence tRNA expression in plants, and in a conditional fashion when bound by inducible proteins.