The use of a real‐time luciferase assay to quantify gene expression dynamics in the living yeast cell

Abstract

A destabilized version of firefly luciferase was used in living yeast cells as a real-time reporter for gene expression. This highly sensitive and non-invasive system can be simultaneously used upon many different experimental conditions in small culture aliquots. This allows the dose-response behaviour of gene expression driven by any yeast promoter to be reported and can be used to quantify important parameters, such as the threshold, sensitivity, response time, maximal activity and synthesis rate for a given stimulus. We applied the luciferase assay to the nutrient-regulated GAL1 promoter and the stress-responsive GRE2 promoter. We find that luciferase expression driven by the GAL1 promoter responds dynamically to growing galactose concentrations, with increasing synthesis rates determined by the light increment in the initial linear phase of activation. In the case of the GRE2 promoter, we demonstrate that the very short-lived version of luciferase used here is an excellent tool to quantitatively describe transient transcriptional activation. The luciferase expression controlled by the GRE2 promoter responds dynamically to a gradual increase of osmotic or oxidative stress stimuli, which is mainly based on the progressive increase of the time the promoter remains active. Finally, we determined the dose-response behaviour of a single transcription factor binding site in a synthetic promoter context, using the stress response element (STRE) as an example. Taken together, the luciferase assay described here is an attractive tool to rapidly and precisely determine and compare kinetic parameters of gene expression.

Deciphering Dynamic Dose Responses of Natural Promoters and Single cis Elements upon Osmotic and Oxidative Stress in Yeast

“…Osmotic and oxidative stresses are harmful conditions for yeast cells and trigger transcriptional programs which include the activation of hundreds of defense genes. We compared the activation patterns of several stress-responsive promoters in yeast in a time-resolved fashion with the help of a destabilized luciferase assay (41). This assay makes use of a very short-lived version of luciferase which is rapidly degraded with the help of specific protein and mRNA degradation motifs.…”

“…Single-copy reporter fusions with destabilized luciferase (lucCP ϩ ) were constructed as described in reference 41. The upstream regulatory sequences of GRE2 (nucleotides Ϫ940 to Ϫ7), CTT1 (nucleotides Ϫ983 to Ϫ10), SOD2 (nucleotides Ϫ977 to Ϫ16), and CCP1 (nucleotides Ϫ976 to Ϫ5) were amplified by PCR and inserted (SacI/SmaI) into the lucCP ϩ expression vector p413-lucCPϩ (41). For the assay of specific cis-regulatory elements, synthetic oligonucleotides containing three repetitions of STRE, CRE, or AP-1 sequences spaced by 8 to 9 nucleotides were inserted into the BspEI site of plasmid p413CYC1⌬-lucCP ϩ (41).…”

“…The upstream regulatory sequences of GRE2 (nucleotides Ϫ940 to Ϫ7), CTT1 (nucleotides Ϫ983 to Ϫ10), SOD2 (nucleotides Ϫ977 to Ϫ16), and CCP1 (nucleotides Ϫ976 to Ϫ5) were amplified by PCR and inserted (SacI/SmaI) into the lucCP ϩ expression vector p413-lucCPϩ (41). For the assay of specific cis-regulatory elements, synthetic oligonucleotides containing three repetitions of STRE, CRE, or AP-1 sequences spaced by 8 to 9 nucleotides were inserted into the BspEI site of plasmid p413CYC1⌬-lucCP ϩ (41). The sequences used were the following (the consensus sequence for each TF binding site is underlined): for STRE, 5=-CCGGCG ATATCAGCCCCTGGAAAAAGCCCCTGCGCAAAGCCCCT-3=; for CRE, 5=-CCGGCGATATCATTACGTAATAGAATACATTACGTAATC GCGATCATTACGTAAT-3=; and for the AP-1 element, 5=-CCGGCATC GATCTTACTAAGCGCGAAATTAGTAACCGGCTAATTACTAAGT-3=.…”

Different Mechanisms Confer Gradual Control and Memory at Nutrient- and Stress-Regulated Genes in Yeast

“…Yeast strains expressing chromosomally tagged TAP fusion proteins were BY4741 (MATa his3⌬1 leu2⌬0 met15⌬0 ura3⌬0) with GAL4-TAP-His3MX and GAL3-TAP-His3MX (36 Plasmid constructions. Single-copy reporter fusions with a destabilized luciferase gene (lucCP ϩ ) were constructed as described previously (38). The upstream regulatory sequences of GRE2 (nucleotides Ϫ940 to Ϫ7), GAL1 (nucleotides Ϫ450 to Ϫ1) (38), CTT1 (nucleotides Ϫ983 to Ϫ10), SOD2 (nucleotides Ϫ977 to Ϫ16) (15), ALD6 (nucleotides Ϫ785 to Ϫ2), and HOR2 (nucleotides Ϫ948 to Ϫ33) (this study) were used.…”

“…Single-copy reporter fusions with a destabilized luciferase gene (lucCP ϩ ) were constructed as described previously (38). The upstream regulatory sequences of GRE2 (nucleotides Ϫ940 to Ϫ7), GAL1 (nucleotides Ϫ450 to Ϫ1) (38), CTT1 (nucleotides Ϫ983 to Ϫ10), SOD2 (nucleotides Ϫ977 to Ϫ16) (15), ALD6 (nucleotides Ϫ785 to Ϫ2), and HOR2 (nucleotides Ϫ948 to Ϫ33) (this study) were used. An integrative version of the GAL1-lucCP ϩ reporter fusion was constructed by insertion of the lucCP ϩ gene into the pAG306GAL1-ccdB Gateway destination vector (39), which was integrated into the URA3 locus of yeast wild-type strain W303-1A.…”