Dataset Information

The upstream activating sequence for L-leucine gene regulation in Saccharomyces cerevisiae.

ABSTRACT: The upstream activating sequence (UAS) conferring leucine-specific regulation of transcription in Saccharomyces cerevisiae was identified by analysis of the LEU2 promoter and by comparison to other genes regulated by leucine. The UAS was localized with deletions and cloned synthetic DNA. Point mutations and sequence rearrangements were used to identify important basepairs and to construct an improved UAS with increased regulation and expression. The improved UAS contains a core ten basepair, GC-rich, palindromic sequence, which is sufficient to confer minimal levels of activation and regulation, within a 36 basepair palindromic sequence which confers maximal activation and regulation. Deletions downstream of the UAS indicated that the UAS must act in conjunction with at least one other site, perhaps a TATAA region, in order to confer high levels of activation. Tandem copies of the UAS in front of LEU2 increased expression and regulation. Tandem UAS elements in trans on a multi-copy 2 mu-based plasmid decreased expression and regulation. These results are consistent with a model that the UAS serves as the DNA-binding site for diffusible activation factor(s), possibly the LEU3 gene product.

SUBMITTER: Tu H

PROVIDER: S-EPMC331095 | biostudies-other | 1990 Jul

REPOSITORIES: biostudies-other

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The upstream activating sequence for L-leucine gene regulation in Saccharomyces cerevisiae.

Tu H H Casadaban M J MJ

Nucleic acids research 19900701 13

The upstream activating sequence (UAS) conferring leucine-specific regulation of transcription in Saccharomyces cerevisiae was identified by analysis of the LEU2 promoter and by comparison to other genes regulated by leucine. The UAS was localized with deletions and cloned synthetic DNA. Point mutations and sequence rearrangements were used to identify important basepairs and to construct an improved UAS with increased regulation and expression. The improved UAS contains a core ten basepair, GC- ...[more]

PMID: 2197599

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Project description:BackgroundConsiderable interest in the bioconversion of lignocellulosic biomass into ethanol has led to metabolic engineering of Saccharomyces cerevisiae for fermentation of xylose. In the present study, the transcriptome and proteome of recombinant, xylose-utilising S. cerevisiae grown in aerobic batch cultures on xylose were compared with those of glucose-grown cells both in glucose repressed and derepressed states. The aim was to study at the genome-wide level how signalling and carbon catabolite repression differ in cells grown on either glucose or xylose. The more detailed knowledge whether xylose is sensed as a fermentable carbon source, capable of catabolite repression like glucose, or is rather recognised as a non-fermentable carbon source is important for further engineering this yeast for more efficient anaerobic fermentation of xylose.ResultsGenes encoding respiratory proteins, proteins of the tricarboxylic acid and glyoxylate cycles, and gluconeogenesis were only partially repressed by xylose, similar to the genes encoding their transcriptional regulators HAP4, CAT8 and SIP1-2 and 4. Several genes that are repressed via the Snf1p/Mig1p-pathway during growth on glucose had higher expression in the cells grown on xylose than in the glucose repressed cells but lower than in the glucose derepressed cells. The observed expression profiles of the transcription repressor RGT1 and its target genes HXT2-3, encoding hexose transporters suggested that extracellular xylose was sensed by the glucose sensors Rgt2p and Snf3p. Proteome analyses revealed distinct patterns in phosphorylation of hexokinase 2, glucokinase and enolase isoenzymes in the xylose- and glucose-grown cells.ConclusionThe results indicate that the metabolism of yeast growing on xylose corresponds neither to that of fully glucose repressed cells nor that of derepressed cells. This may be one of the major reasons for the suboptimal fermentation of xylose by recombinant S. cerevisiae strains. Phosphorylation of different isoforms of glycolytic enzymes suggests that regulation of glycolysis also occurred at a post-translational level, supporting prior findings.

Dataset Information

The upstream activating sequence for L-leucine gene regulation in Saccharomyces cerevisiae.

Publications

The upstream activating sequence for L-leucine gene regulation in Saccharomyces cerevisiae.

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