A modified yeast three-hybrid system enabling both positive and negative selections

Wallis, Christopher P; Filipovska, Aleksandra; Rackham, Oliver

doi:10.1007/s10529-018-2567-7

Cited by 3 publications

(2 citation statements)

References 21 publications

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“…A series of GAL4 variants were used that combined strong or weak Kozak consensus sequences and start codons to further modulate the amount of Gal4 produced (Figure a). We coupled these tetracycline-responsive Gal4 expression plasmids with a yeast strain harboring HIS3 and URA3 biosynthetic genes under control of Gal4-inducible upstream activating sequence (UAS GAL ) elements, to enable growth in media lacking histidine and uracil, respectively, if Gal4 is present (Figure b). , Using URA3 as a reporter gene also provided a means for negative selection, as the enzyme encoded by URA3 will decarboxylate 5-fluoroorotic acid (5-FOA) to yield the toxic metabolite 5-fluorouracil (5-FU) (Figure c). Furthermore, the competitive inhibitor of the protein product of HIS3 , 3-amino-1,2,4-triazole (3-AT), can be used to eliminate false positives that arise from uninduced leaky HIS3 expression in the absence of ATC (Figure c).…”

Section: Resultsmentioning

confidence: 99%

An Artificial Yeast Genetic Circuit Enables Deep Mutational Scanning of an Antimicrobial Resistance Protein

et al. 2018

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Understanding the molecular mechanisms underlying antibiotic resistance requires concerted efforts in enzymology and medicinal chemistry. Here we describe a new synthetic biology approach to antibiotic development, where the presence of tetracycline antibiotics is linked to a life-death selection in Saccharomyces cerevisiae. This artificial genetic circuit allowed the deep mutational scanning of the tetracycline inactivating enzyme TetX, revealing key functional residues. We used both positive and negative selections to confirm the importance of different residues for TetX activity, and profiled activity hotspots for different tetracyclines to reveal substrate-specific activity determinants. We found that precise positioning of FAD and hydrophobic shielding of the tetracycline are critical for enzymatic inactivation of doxycycline. However, positioning of FAD is suboptimal in the case of anhydrotetracycline, potentially explaining its comparatively poor degradation and potential as an inhibitor for this family of enzymes. By combining artificial genetic circuits whose function can be modulated by antimicrobial resistance determinants, we establish a framework to select for the next generation of antibiotics.

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Section: Resultsmentioning

confidence: 99%

An Artificial Yeast Genetic Circuit Enables Deep Mutational Scanning of an Antimicrobial Resistance Protein

et al. 2018

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“…Fortunately, yeast strains already engineered to assay macromolecular interactions (for example yeast two-hybrid systems) can be appropriated for part of this genetic circuitry (Fields & Song, 1989;Licitra & Liu, 1996;Van Criekinge & Beyaert, 1999;Wallis, Filipovska, & Rackham, 2018). In these strains, when an experimental macromolecular interaction occurs, a functional transcription factor is created that activates the transcription of a reporter gene.…”

Section: General Principlesmentioning

confidence: 99%

Building artificial genetic circuits to understand protein function

Scott

Mathews

Filipovska

et al. 2020

Methods in Enzymology

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Intrinsic protein properties that may not be apparent by only examining three-dimensional structures can be revealed by careful analysis of mutant protein variants. Deep mutational scanning is a technique that allows the functional analysis of millions of protein variants in a single experiment. To enable this high-throughput technique, the mutant genotype of protein variants must be coupled to a selectable function. This chapter outlines how artificial genetic circuits in the yeast Saccharomyces cerevisiae can maintain the genotype-phenotype link, thus enabling the general application of this approach. To do this, we describe how to engineer genetic selections in yeast, methods to construct mutant libraries, and how to analyze sequencing data. We investigate the structure-function relationships of the antimicrobial resistance protein TetX to illustrate this process. In doing so, we demonstrate that deep mutational scanning is a powerful method to dissect the importance of individual residues for the inactivation of antibiotics analogues, with consequences for the rational design of new drugs to combat antimicrobial resistance.

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rec-Y3H screening allows the detection of simultaneous RNA-protein interface mutations

Garriga-Canut

Yang

Preußer

et al. 2020

Methods

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A modified yeast three-hybrid system enabling both positive and negative selections

Abstract: This yeast three-hybrid system has advantages over previous versions as demonstrated by the increased dynamic range of detectable binding interactions using yeast survival assays and colony forming assays with multiple reporters using known RNA-protein interactions.

Cited by 3 publications

References 21 publications

An Artificial Yeast Genetic Circuit Enables Deep Mutational Scanning of an Antimicrobial Resistance Protein

An Artificial Yeast Genetic Circuit Enables Deep Mutational Scanning of an Antimicrobial Resistance Protein

Building artificial genetic circuits to understand protein function

rec-Y3H screening allows the detection of simultaneous RNA-protein interface mutations

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