The use of a real-time luciferase assay to quantify gene expression dynamics in the living yeast cell.
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.
Instituto de Biología Molecular y Celular de Plantas, CSIC-Universidad Politécnica de Valencia, Ciudad Politécnica de la Innovación, Valencia, Spain.
Alessandro
A
Rienzo
Amparo
A
Pascual-Ahuir
Markus
M
Proft
D019677:Artificial Gene Fusion; D005690:Galactose / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D017930:Genes, Reporter; D005827:Genetics, Microbial / Q000379:methods*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D009997:Osmotic Pressure; D018384:Oxidative Stress; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
D008156:Luciferases; D005690:Galactose