Improvement of the reverse tetracycline transactivator by single amino acid substitutions that reduce leaky target gene expression to undetectable levels.
Conditional gene expression systems that enable inducible and reversible transcriptional control are essential research tools and have broad applications in biomedicine and biotechnology. The reverse tetracycline transcriptional activator is a canonical system for engineered gene expression control that enables graded and gratuitous modulation of target gene transcription in eukaryotes from yeast to human cell lines and transgenic animals. However, the system has a tendency to activate transcription even in the absence of tetracycline and this leaky target gene expression impedes its use. Here, we identify single amino-acid substitutions that greatly enhance the dynamic range of the system in yeast by reducing leaky transcription to undetectable levels while retaining high expression capacity in the presence of inducer. While the mutations increase the inducer concentration required for full induction, additional sensitivity-enhancing mutations can compensate for this effect and confer a high degree of robustness to the system. The novel transactivator variants will be useful in applications where tight and tunable regulation of gene expression is paramount.
Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, K1H8L1, Canada.|Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, K1H8L1, Canada.
Ian J
IJ
Roney
Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, K1H8L1, Canada.|Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, K1H8L1, Canada.
Adam D
AD
Rudner
Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, K1H8L1, Canada.|Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, K1H8L1, Canada.
Jean-François
JF
Couture
Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, K1H8L1, Canada.|Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, K1H8L1, Canada.|Department of Physics, University of Ottawa, Ottawa, K1N6N5, Canada.|Cancer Therapeutics Program, Ottawa Hospital Research Institute, Ottawa, K1H8L6, Canada.
Mads
M
Kærn
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D030801:Animals, Genetically Modified; D001709:Biotechnology / Q000379:methods*; D002460:Cell Line; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D006801:Humans; D013752:Tetracycline / Q000378:metabolism* / Q000494:pharmacology; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics*; D015003:Yeasts / Q000235:genetics
D015534:Trans-Activators; D013752:Tetracycline