An improved short-lived fluorescent protein transcriptional reporter for Saccharomyces cerevisiae

Abstract

Ideal reporter genes for temporal transcription programmes have short half-lives that restrict their detection to the window in which their transcripts are present and translated. In an effort to meet this criterion for reporters of transcription in individual living cells, we adapted the ubiquitin fusion strategy for programmable N-end rule degradation to generate an N-degron version of green fluorescent protein (GFP) with a half-life of ~7 min. The GFP variant we used here (designated GFP*) has excellent fluorescence brightness and maturation properties, which make the destabilized reporter well suited for tracking the induction and attenuation kinetics of gene expression in living cells. These attributes are illustrated by its ability to track galactose- and pheromone-induced transcription in S. cerevisiae. We further show that the fluorescence measurements using the short-lived N-degron GFP* reporter gene accurately predict the transient mRNA profile of the prototypical pheromone-induced FUS1 gene.

Figure 1

Strategy for N-degron GFP* reporter gene construction and integration into the S. cerevisiae genome. Plasmids with different UAS-UBI-X-ΔkGFP* reporters are constructed by combining UAS and GFP targeting cassettes. The X-Δk element of the reporter gene represents different bipartite N-degron signal sequences (see text). Digestion of the reporter gene plasmid with SacI and SalI restriction endonucleases releases the reporter gene from the plasmid backbone and generates free ends that target the allele to the URA3-TIM9 intergenic region of chromosome V. Unique restriction enzyme sites in the different cassettes are shown (B, BamHI; G, BglII; E, EcoRV; K, KpnI; Sa, SalI; Sc, SacI; Sm, SmaI; X, XhoI.)

Figure 2

Galactose induction kinetics for detection of reporter gene mRNA, protein, and fluorescence. (A & B) Plots comparing galactose induction profiles for GFP* mRNA (), protein (), and fluorescence () in strains with the ^PGAL1-UBI-MΔkGFP* (C699-181) or ^PGAL1-UBI-YΔkGFP* (C699-183) reporters, as specified. The data points for mRNA and protein are the average values from four and three independent induction time courses, respectively. The data points for fluorescence are the average of the mean fluorescence from four independent induction time courses. (The mean fluorescence value at each time in given time course is determined from quantification of at least 25 cells.) Error bars show 95% confidence limits calculated from the independent time course determinations.

Figure 3

Degradation kinetics of reporter gene mRNA, protein, and fluorescence. (A & B) Plots show the exponential decay of GFP* mRNA () and protein quantified by Western blot () or fluorescence () in strains with the GAL1-UBI-MΔkGFP* (C699-181) or GAL1-UBI-YΔkGFP* (C699-183) reporters, as specified. Amounts of each species are relative to steady state amounts before inhibition of transcription (t=0) by the addition of glucose to the cultures. Data points shown in each plot are the average of N independent experiments as specified in Table 3. Error bars show 95% confidence limits.

Figure 4

Comparison of pheromone induction profiles for FUS1 and reporter gene expression. (A) Plot comparing pheromone induction profiles for FUS1 () and GFP* () mRNA in ^PFUS1-UBI-MΔkGFP* (C699-198) and ^PFUS1-UBI-YΔkGFP* (C699-199) strains. Data points are the average from six independent induction time courses (three with strain C699-198 and three with strain C699-199). Error bars show 95% confidence limits. (B) Plot comparing the pheromone induction profile for GFP* fluorescence in the ^PFUS1-UBI-MΔkGFP* (C699-198) () and ^PFUS1-UBI-YΔkGFP* (C699-199) reporter strains. More than 25 or 50 individual cells were scored for each time point in a single time course with strain C699-198 or C699-199, respectively. Each data point is the average from four independent time courses. Error bars show 95% confidence limits. (C) Model prediction of the pheromone induced FUS1 mRNA profile using YΔkGFP* fluorescence measurements as input. Plot compares the model mean () and range (min, max ) to the empirically determined reporter GFP* mRNA profile from (A).