Improvement of the reverse tetracycline transactivator by single amino acid substitutions that reduce leaky target gene expression to undetectable levels.

Mads Kærn,Ian J. Roney,Adam D. Rudner,Jean-François Couture

doi:10.1038/srep27697

Mads Kærn, Ian J. Roney + Show 2 more

Open Access

https://doi.org/10.1038/srep27697

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Journal: Scientific reports	Publication Date: Jun 21, 2016
Citations: 56	License type: CC BY 4.0

Affiliation: University of Ottawa, Ottawa Hospital

Abstract

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.

Highlights

The reverse tetracycline transactivator (rtTA)/PTET-yeast-enhanced green fluorescent protein (yeGFP) expression cassette was integrated in a single copy into the yeast genome
We have identified single amino acid substitutions in the widely used doxycycline-inducible transactivator that significantly improves dynamic range without compromising maximal expression capacity
Replacing a single glycine (G72) in the rtTA-M2 variant with residues that introduce non-polar side chains reduces its ability to activate transcription in the absence of doxycycline in a manner that depends on the size of the side chain

Summary

Introduction

To use rtTA in this context, we created two strong doxycycline-responsive promoters with either three (PTET3) or four (PTET4) rtTA binding sites, and used the optimized rtTA-M2 variant[7] to control the expression of yeast-enhanced green fluorescent protein (yeGFP)[23] from these promoters (Fig. 1a). Despite this improvement, both flow cytometry data (Fig. 1c) and fluorescence microscopy data (Fig. 1d) indicated that un-induced rtTA-M2 causes significant reporter gene expression even when the transactivator is expressed from a weak promoter.

Results

Conclusion