There are two enolase genes, ENO] and EN02, per haploid yeast genome. Expression of the ENO] gene is quantitatively similar in cells grown on glucose or gluconeogenic carbon sources. In contrast, EN02 expression is induced more than 20-fold in cells grown on glucose as the carbon source. cis-Acting regulatory sequences were mapped within the 5'-flanking region of the constitutively expressed yeast enolase gene ENO]. A complex positive regulatory region was located 445 base pairs (bp) upstream from the transcriptional initiation site which was required for ENO] expression in cells grown on glycolytic or gluconeogenic carbon sources. A negative regulatory region was located 160 bp upstream from the transcriptional initiation site. Sequences required for the function of this negative regulatory element were mapped to a 38-bp region. Deletion of all or a portion of these latter sequences permitted glucose-dependent induction of ENO] expression that was quantitatively similar to that of the glucose-inducible EN02 gene. The negative regulatory element therefore prevents glucose-dependent induction of the ENO] gene. Hybrid 5'-flanking regions were constructed which contained the upstream regulatory sequences of one enolase gene fused at a site upstream from the TATAAA box in the other enolase gene. Analysis of the expression of enolase genes containing these hybrid 5'-flanking region showed that the positive regulatory regions of ENO] and EN02 were functionally similar, as were the regions extending from the TATAAA boxes to the initiation codons. Based on these studies, we conclude that the negative regulatory element plays the critical role in maintaining the constitutive expression of the ENO] structural gene in cells grown on glucose or gluconeogenic carbon sources.Enolase is one of the most abundant enzymes in Saccharomyces cerevisiae. There are two yeast enolase structural genes, designated ENO] and EN02, which encode polypeptides differing in 20 of 436 amino acid residues (3). The two genes are expressed differentially in vegetative cells grown on glycolytic or gluconeogenic carbon sources. The steady-state concentrations of the ENOJ-encoded mRNA and polypeptide are similar in cells grown on the two carbon sources, whereas the intracellular concentrations of the ENO2-encoded mRNA and polypeptide are more than 20-fold higher in cells grown on glucose than on glycerol plus lactate (9). We showed previously that transcription of the ENO2 gene is regulated by upstream activation sequences located approximately 460 base pairs (bp) upstream from the transcription initiation site (2). Genetic analysis further showed that sequences within this regulatory region mediate the observed glucose-dependent induction of transcription of ENO2 (2).Having located the cis-acting sequences which regulate transcription of the ENO2 structural gene, we were interested to determine how transcription of the constitutively expressed ENO] structural gene is regulated. Of particular interest is the issue of coordinate regulation of transcri...

The intracellular concentrations of the polypeptides encoded by the two enolase (ENO] and ENO2) and three glyceraldehyde-3-phosphate dehydrogenase (TDHI, TDH2, and TDH3) genes were coordinately reduced more than 20-fold in a Saccharomyces cerevisiae strain carrying the gcrl-l mutation. The steady-state concentration of glyceraldehyde-3-phosphate dehydrogenase mRNA was shown to be approximately 50-fold reduced in the mutant strain. Overexpression of enolase and glyceraldehyde-3-phosphate dehydrogenase in strains carrying multiple copies of either ENO] or TDH3 was reduced more than 50-fold in strains carrying the gcrl -1 mutation. These results demonstrated that the GCRI gene encodes a trans-acting factor which is required for efficient and coordinate expression of these glycolytic gene families. The GCRI gene and the gcrl -I mutant allele were cloned and sequenced. GCRI encodes a predicted 844-amino-acid polypeptide; the gcrl-1 allele contains a 1-base-pair insertion mutation at codon 304. A null mutant carrying a deletion of 90% of the GCRI coding sequence and a URA3 gene insertion was constructed by gene replacement. The phenotype of a strain carrying this null mutation was identical to that of the gcrl-l mutant strain.Yeast glycolytic enzymes make up 25 to 60% of the soluble protein in the organism (7). In most strains of the yeast Saccharomyces cerevisiae, the intracellular concentrations of glycolytic enzymes remain constant in cells grown on glucose or gluconeogenic carbon sources. A notable exception is the enzyme encoded by one of the enolase genes (ENO2), which is induced more than 20-fold in cells grown in glucose (12). In some yeast strains, as well as under certain growth conditions, it has been reported that several other glycolytic enzyme activities are induced when cells are shifted from gluconeogenic carbon sources to glucose (11). This paradox could be explained if many glycolytic genes are capable of glucose-dependent induction only under certain defined physiological conditions or in certain defined genetic backgrounds. Both positive and negative cis-acting regulatory sequences have been identified within the 5' flanking region of the enolase genes (4; R. Cohen, T. Yokoi, J. P. Holland, and M. J. Holland, submitted for publication). By analogy with other yeast genes (1, 6), regulation of glycolytic gene expression by cis-acting sequences is likely to be mediated by trans-acting regulatory proteins.Clifton et al. (3) reported the isolation of a recessive mutation designated gcrl-l. The specific activities of many glycolytic enzymes, including glyceraldehyde-3-phosphate dehydrogenase and enolase, are reduced to 2 to 5% of the wild-type level in strains carrying the gcrl-J mutation. In vitro translational studies suggest that this mutation affects the intracellular levels of glycolytic mRNAs (2). The GCRI gene, therefore, appears to encode a positive transcription factor which is involved in coordinate expression of glycolytic genes. * Corresponding author.In this report, we describe the effects...