Multiple forms of alcohol dehydrogenase in Saccharomyces cerevisiae

Transcription of the ADH2 gene in the yeast Saccharomyces cerevisiae was inhibited by excess copies of its own promoter region. This competition effect was promoter specific and required the upstream activation sequence of ADH2 as well as sequences 3' to the TATA box. Introducing excess copies of ADRI, an ADH2-specific regulatory gene, did not alleviate the competition that was observed in these circumstances during both constitutive and derepressed ADH2 expression. Excess copies of the upstream region did not release ADH2 from glucose repression, consistent with the view that ADH2 is regulated by positive trans-acting factors.Transcriptional control of eucaryotic gene expression by RNA polymerase II is mediated by the binding of regulatory proteins to upstream activator (or operator) DNA sequences, usually located upstream of the TATA box (16). Binding of repressor proteins to operator sequences plays a major role in negatively regulating early transcription in simian virus 40 (31) and in regulating the mating type of Saccharomyces cerevisiae (24). However, initiation of transcription from most known eucaryotic promoters is positively regulated by the binding of specific transcriptional activator proteins to the enhancer region or upstream activation sequence (UAS) of a promoter (9,10,15, 17,20,22,26,35,36,39). These regulatory elements are usually short (6-to 8-base-pair [bp]) repetitive DNA sequences or a single longer sequence which can function in either orientation and at variable distances from a TATA box (16). The TATA box itself has been shown to be the binding site for transcription factor IID, a more general type of transcription factor for RNA polymerase II in mammals (39) and in Drosophila melanogaster (34). These DNA-binding proteins are involved in the formation of a stable complex for initiation of transcription, along with RNA polymerase II and at least two general factors (10, 39).In eucaryotic organisms as well as in bacteria, titration of limiting amounts of regulatory factors in vivo has been useful for identifying and characterizing protein-binding sites on DNA which regulate the activity of promoters. In principle, this experimental approach allows positive versus negative control of gene expression to be distinguished. Competitive binding of an activator protein by excess regulatory DNA sites would decrease gene expression, whereas titration of a repressor protein would increase gene expression. Competition experiments also allow one to examine the effect of regulatory site mutations on the binding of regulatory factors independently of the effect of these mutations on gene expression in cis. In Escherichia coli, both the gal and lac operons are induced when their respective repressor molecules are bound quantitatively to their own operator DNA carried on high-copy-number plasmids or on another genome (23,33

mentioning

confidence: 99%

Transcription of the ADH2 gene in Saccharomyces cerevisiae is limited by positive factors that bind competitively to its intact promoter region on multicopy plasmids.

Irani¹,

Taylor²,

Young³

1987

“…ADH I, the classical fermentative isozyme, is responsible for the last step in the yeast glycolytic pathway, the reduction of acetaldehyde to ethanol (23,28). ADH II, the oxidative isozyme, is highly repressed by fermentative growth and is derepressed in the absence of a fermentable sugar such as glucose.…”

mentioning

confidence: 99%

Isolation and DNA sequence of ADH3, a nuclear gene encoding the mitochondrial isozyme of alcohol dehydrogenase in Saccharomyces cerevisiae.

Young¹,

Pilgrim²

1985

134

113

The Saccharomyces cerevisiae nuclear gene, ADH3, that encodes the mitochondrial alcohol dehydrogenase isozyme ADH III was cloned by virtue of its nucleotide homology to ADHI and ADH2. Both chromosomal and plasmid-encoded ADH III isozymes were repressed by glucose and migrated heterogeneously on nondenaturing gels. Nucleotide sequence analysis indicated 73 and 74% identity for ADH3 with ADHI and ADH2, respectively. The amino acid identity between the predicted ADH III polypeptide and ADH I and ADH II was 79 and 80%, respectively. The open reading frame encoding ADH III has a highly liasic 27-amino-acid amino-terminal extension relative to ADH I and ADH II. The nucleotide sequence of the presumed leader peptide has a high degree of identity with the untranslated leader regions ofADHI and ADH2 mRNAs. A strain containing a null allele of ADH3 did not have a detectably altered phenotype. The cloned gene integrated at the ADH3 locus, indicating that this is the structural gene for ADH III.The alcohol dehydrogenase (ADH) isozymes of Saccharomyces cerevisiae represent a functionally diverse enzyme family. ADH I, the classical fermentative isozyme, is responsible for the last step in the yeast glycolytic pathway, the reduction of acetaldehyde to ethanol (23,28). ADH II, the oxidative isozyme, is highly repressed by fermentative growth and is derepressed in the absence of a fermentable sugar such as glucose. ADH2, the structural gene for ADH II, is regulated at the transcriptional level by catabolite repression through a specific positive effector encoded by the ADRI gene (8, 10). The function of ADH II in the cell is to oxidize ethanol, formed during fermentation, to acetaldehyde, which can then be metabolized via the tricarboxylic cycle in the mitochondria and also serves as an intermediate in gluconeogenesis.ADH III was discovered by Lutsdorf and Megnet (23) and was shown to purify with a particulate fraction from S. cerevisiae. Sugar et al. (35) further characterized this activity and confirmed its location in the mitochondrion. Wills and Phelps (45) obtained evidence that the size of the native enzyme was the same as that of ADH I. Despite the same apparent molecular weight, the native ADH III enzyme migrates in an unusual pattern on nondenaturing polyacrylamide gels, suggesting charge heterogeneity on the subunits or formation of heterotetramers synthesized by two different genes or both.The metabolic function of ADH III is unknown, but indications are that it does not function fermentatively: strains containing ADH III as their only ADH isozyme cannot survive as petites, which must obtain their energy through fermentation (45). A role in respiratory metabolism is thus suggested, which would be consistent with a mitochondrial location. Molecular cloning of the structural gene for ADH III would aid in elucidating its structure and function. The structural genes encoding ADH I and ADH II, ADHI and ADH2, respectively, have been cloned and sequenced (3,30,41,42), allowing comparisons to be made * Corresponding author. ...

“…Neither ADH II enzyme activity nor its mRNA is detectable when S. cerevisiae is grown on a fermentable carbon source such as glucose (11,21). The enzyme is derepressed over 100-fold when the fermentable substrate in the medium is exhausted or replaced by a nonfermentable carbon source such as glycerol or ethanol.…”

mentioning

confidence: 99%

Deletion analysis identifies a region, upstream of the ADH2 gene of Saccharomyces cerevisiae, which is required for ADR1-mediated derepression.

Beier¹,

Sledziewski²,

Young³

1985

Deletion analysis was used to identify sequences upstream of the ADH2 gene of Saccharomyces cerevisiae that are required for its regulation. S' and 3' internal deletions of the ADH2 control region were created in vitro, and the fragments were ligated adjacent to the ADHI promoter and structural gene. Hybrid genes with 3' deletions extending from -119 to -216 (the start site of ADH2 transcription is designated + 1) were fully repressed and derepressed to high levels. Hybrid genes with 3' deletions extending from -119 to -257 were repressed but failed to significantly derepress. Hybrid genes lacking the -216 to -257 region also failed to respond to ADRI-5c, a mutant allele of the unlinked regulatory gene ADRI, which confers constitutive expression on ADH2. This implies that the region between these deletion endpoints, which includes a 22-base-pair sequence of dyad symmetry, is required for efficient derepression of an adjacent promoter. Internal deletions extending in the 3' direction from position -1141 confirmed these results. Deletion mutants lacking the region -1141 to -259 were normally regulated, whereas deletions extending from -1141 to -115 were not derepressible. These results support the hypotheses that the ADH2 promoter may normally be in an inactive conformation in the yeast chromosome and that derepression of ADH2 requires positive activation mediated through an upstream activation sequence located between 216 and 257 base pairs 5' to the start site of ADH2 transcription. No evidence for a DNA sequence mediating repression was obtained.