Positive and negative regulation of squalene synthase (ERG9), an ergosterol biosynthetic gene, in Saccharomyces cerevisiae.
To identify regulatory cis-elements in the proximal promoter of the yeast ERG9 squalene synthase gene, promoter deletion analysis was performed. This approach identified two regulatory elements, one an upstream repressing cis-element (URS), and the other an upstream activating cis-element (UAS). Electromobility shift assays (EMSAs) demonstrated that distinct proteins bind each element. Genetic screens were performed to identify yeast mutants that altered expression of ERG9 promoter-reporter gene fusions. Three non-ergosterol biosynthetic pathway genes were identified. A mutation in TPO1(YLL028W) led to a 5.5-fold increase in ERG9 expression while mutations in YER064C and SLK19 (YOR195W) led to a 3.1- and 5.6-fold decrease, respectively. Deletion analysis of these genes demonstrated that TPO1 and SLK19 specifically regulated ERG9 expression when tested with several different promoter-reporter gene fusions. Additionally, EMSAs demonstrated that extracts derived from the TPO1 deletion strain was unable to shift the repressing cis-element while protein extracts from the SLK19 deletion strain had a reduced shift of the activating cis-element. Furthermore, these two mutants showed quantitative differences in sterols and antifungal drug susceptibilities consistent with their role in regulating ERG9 expression.
Kennedy
M A
MA
Department of Biology, Indiana University Purdue University, 723 West Michigan Street, Indianapolis, IN 46202, USA.
Bard
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M
D001483:Base Sequence; D004875:Ergosterol / Q000096:biosynthesis*; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000096:biosynthesis / Q000235:genetics*; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic
D013186:Farnesyl-Diphosphate Farnesyltransferase; D004875:Ergosterol