title: 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.
authors.affiliation: Department of Physics, University of North Carolina, Chapel Hill, NC 27599-7260, USA.
authors.forename: John R
authors.initials: JR
authors.lastname: Houser
authors.forename: Eintou
authors.initials: E
authors.lastname: Ford
authors.forename: Sudeshna M
authors.initials: SM
authors.lastname: Chatterjea
authors.forename: Seth
authors.initials: S
authors.lastname: Maleri
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Elston
authors.forename: Beverly
authors.initials: B
authors.lastname: Errede
mesh_terms: D005690:Galactose / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006207:Half-Life; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics; D010675:Pheromones / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012331:RNA, Fungal / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: C490024:FUS1 protein, S cerevisiae; D008565:Membrane Proteins; D010675:Pheromones; D012331:RNA, Fungal; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D005690:Galactose