title: Comprehensive Analysis of the SUL1 Promoter of Saccharomyces cerevisiae.
abstract: In the yeast Saccharomyces cerevisiae, beneficial mutations selected during sulfate-limited growth are typically amplifications of the SUL1 gene, which encodes the high-affinity sulfate transporter, resulting in fitness increases of >35% . Cis-regulatory mutations have not been observed at this locus; however, it is not clear whether this absence is due to a low mutation rate such that these mutations do not arise, or they arise but have limited fitness effects relative to those of amplification. To address this question directly, we assayed the fitness effects of nearly all possible point mutations in a 493-base segment of the gene's promoter through mutagenesis and selection. While most mutations were either neutral or detrimental during sulfate-limited growth, eight mutations increased fitness >5% and as much as 9.4%. Combinations of these beneficial mutations increased fitness only up to 11%. Thus, in the case of SUL1, promoter mutations could not induce a fitness increase similar to that of gene amplification. Using these data, we identified functionally important regions of the SUL1 promoter and analyzed three sites that correspond to potential binding sites for the transcription factors Met32 and Cbf1 Mutations that create new Met32- or Cbf1-binding sites also increased fitness. Some mutations in the untranslated region of the SUL1 transcript decreased fitness, likely due to the formation of inhibitory upstream open reading frames. Our methodology-saturation mutagenesis, chemostat selection, and DNA sequencing to track variants-should be a broadly applicable approach.
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington 98195 Synthetic Biology Division, Research Center for Advanced Science and Technology, the University of Tokyo, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Rich
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington 98195.
authors.forename: Celia
authors.initials: C
authors.lastname: Payen
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington 98195 Bioinformatics Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, Victoria 3052, Australia Bioinformatics and Cancer Genomics Laboratory, Peter MacCallum Cancer Centre, Victoria 3002, Australia Department of Medical Biology, University of Melbourne, Victoria 3010, Australia.
authors.forename: Alan F
authors.initials: AF
authors.lastname: Rubin
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington 98195.
authors.forename: Giang T
authors.initials: GT
authors.lastname: Ong
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington 98195.
authors.forename: Monica R
authors.initials: MR
authors.lastname: Sanchez
authors.affiliation: Synthetic Biology Division, Research Center for Advanced Science and Technology, the University of Tokyo, Meguro-ku, Tokyo 153-8904, Japan Institute for Advanced Bioscience, Keio University, Tsuruoka, Yamagata 997-0035, Japan PRESTO, Japan Science and Technology Agency, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Nozomu
authors.initials: N
authors.lastname: Yachie
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington 98195.
authors.forename: Maitreya J
authors.identifier: 0000-0001-9944-2666
authors.initials: MJ
authors.lastname: Dunham
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington 98195 Department of Medicine, University of Washington, Seattle, Washington 98195 Howard Hughes Medical Institute, University of Washington Seattle, Washington 98195 fields@uw.edu.
authors.forename: Stanley
authors.initials: S
authors.lastname: Fields
mesh_terms: D027321:Anion Transport Proteins / Q000235:genetics*; D001665:Binding Sites; D015966:Gene Expression Regulation, Fungal; D056084:Genetic Fitness; D016296:Mutagenesis; D009154:Mutation; D059372:Nucleotide Motifs; D016366:Open Reading Frames; D056510:Position-Specific Scoring Matrices; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D000076988:Sulfate Transporters; D014157:Transcription Factors
chemical_list: D027321:Anion Transport Proteins; C094656:SUL1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000076988:Sulfate Transporters; D014157:Transcription Factors
keywords: Saccharomyces cerevisiae; chemostat; gene amplification; high-throughput sequencing; promoter mutagenesis; transcription; yeast