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 tRNA(Lys)AUC or tRNA(Trp)AUC 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 tRNA(Trp)AUC gene), or at positions -2, -6 and -10 (for the tRNA(Lys)AUC gene), and repression reached 90%. The inducer tetracycline (Tc) restored tRNA expression. Similarly, carrot protoplasts transfected with human tRNA(Ser)AUC 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 occurred by the lactose repressor when lacO was located at position -1 of the tRNA(Ser)AUC coding sequence. In the presence of the inducer isopropyl-beta-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.
Ulmasov
B
B
Department of Biochemistry, University of Missouri-Columbia 65211, USA.
Capone
J
J
Folk
W
W
D017360:Arabidopsis / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D018552:Daucus carota; D029968:Escherichia coli Proteins; D018506:Gene Expression Regulation, Plant / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D056968:Lac Repressors; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic / Q000235:genetics; D011387:Prokaryotic Cells; D014176:Protein Biosynthesis / Q000235:genetics; D011523:Protoplasts; D012343:RNA, Transfer / Q000235:genetics*; D012357:RNA, Transfer, Lys / Q000235:genetics; D012362:RNA, Transfer, Ser / Q000235:genetics; D012364:RNA, Transfer, Trp / Q000235:genetics; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins / Q000235:genetics / Q000502:physiology*; D013489:Suppression, Genetic / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D012357:RNA, Transfer, Lys; D012362:RNA, Transfer, Ser; D012364:RNA, Transfer, Trp; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D012343:RNA, Transfer; D013752:Tetracycline