Sequences within an upstream activation site in the yeast enolase gene ENO2 modulate repression of ENO2 expression in strains carrying a null mutation in the positive regulatory gene GCR1.

Abstract

Transcription of the yeast enolase gene EN02 is reduced 20-to 50-fold in strains carrying a null mutation in the positive regulatory gene GCRI. A small deletion mutation within one of two upstream activation sites (UAS elements) in the 5'-flanking region of EN02 permitted wild-type levels of EN02 gene expression in a strain carrying the gcrl null mutation. These data show that sequences required for UAS element activity in GCRI strains were required to repress EN02 expression in a gcrl strain. Protein factors that specifically bound to this UAS/repression site were identified. We show that the DNA-binding protein ABFI (autonomously replicating sequence-binding factor) is the major protein which binds the UAS/repression site. Minor DNA-binding activities that interact specffically with the UAS/repression site were also identified and may correspond to proteolytic breakdown products of ABFI. None of the observed binding activities were encoded by the GCRI structural gene. A double-stranded oligonucleotide that included the UAS/repression site activated transcription of UAS-less ENO] and EN02 gene cassettes in vivo to wild-type levels in strains carrying the GCRI allele as well as the geri null mutation. These latter data show that the UAS/repression site is sufficient for transcriptional activation but is not sufficient to repress transcription of the enolase genes in a gcrl genetic background.Maximal expression of most glycolytic genes in Saccharomyces cerevisiae requires the GCRI gene product. The specific activities of many glycolytic enzymes are reduced 5-to 50-fold in strains carrying a gcrl null mutation (5, 6, 10). Transcription of the two enolase genes, ENOI and EN02, as well as all three glyceraldehyde-3-phosphate dehydrogenase genes is reduced 20-to 50-fold in a strain carrying a gcrl null mutation (10). These results suggest that the GCRI gene product is a positive regulator of glycolytic gene expression. Although the GCRI gene is not essential for cell viability, cells carrying a geri null mutation grow poorly on a medium containing glucose as the carbon source. In contrast, GCRI and gcrl mutant strains grow at similar rates on gluconeogenic carbon sources, such as glycerol plus lactate. The GCRJ structural gene has been cloned and sequenced (1, 10); however, the biological activity of the GCRJ gene product is unknown. cis-Acting regulatory sequences which are required for expression of the two yeast enolase genes have been mapped within the 5'-flanking regions of each respective gene (7,8,(18)(19)(20). Since the GCRJ gene product appears to be a positive activator of transcription of the enolase genes, it was of interest to identify the cis-acting sequences which mediate the activity of this protein.In this report we show that cis-acting sequences that are part of an upstream activation site (UAS) capable of activating EN02 expression in a strain carrying a wild-type GCRI gene modulated repression of EN02 expression in a strain carrying a gcri null mutation. DNA-binding proteins that interact specific...

A complex regulatory element from the yeast gene ENO2 modulates GCR1-dependent transcriptional activation.

“…These observations are consistent with earlier studies showing that the presence of the ABFI-or RAP1-binding site is required for EN02 expression (17). As observed previously for a small deletion mutation that removed the ABFI-binding site (31), the MtoLl (-486 to -390, abf-) fragment containing base substitution mutations that abolish ABFI binding displayed high levels of UAS activity in a strain carrying the gcrl-l null mutation.…”

“…We showed previously that the overlapping ABFI and RAP1 sites alone (E2u; -486 to -447) conferred near-wild-type levels of gene expression when ligated into the Sall site of plasmid penoF26; however, expression was not dependent on the GCR1 gene product (31). In marked contrast, EN02 sequences extending from positions -486 to -390 (Fig.…”

“…Conversely, the GCR1-binding site in ENO1 exhibited no UAS activity when base substitution mutations that greatly reduced RAP1 binding were introduced (10). These Previous studies of the EN02 5' regulatory sequences identified binding sites of ABFI and RAP1 between positions -486 and -450 (7,31). Deletion-mapping analysis showed that EN02 genes containing a binding site for either ABFI or RAP1 were expressed at near-wild-type levels in a GCR1 strain, whereas removal of both binding sites abolished EN02 transcription (17).…”

“…Regulatory sequences from the 5'-flanking region of the EN02 gene were amplified by PCR (37). For Cells were harvested at an A660 of 1.0 and disrupted with an Eaton press, and an S100 whole-cell extract was prepared as previously described (31). S100 whole-cell extract (6.4 mg of protein) was fractionated by Mono S chromatography as previously described (7).…”

A simplein vivofootprinting method to examine DNA-protein interactions over the yeastPYKUAS element

“…The GCRI gene product is a positive activator required for the expression of many yeast glycolytic genes (25 -28). Studies indicate that this activator effects uene expression at the level of transcription (25,28). To demonstrate the requirement for the GCR 1 trains-activating factor, PYK gene expression studies were carried out in wild-type and gcrl null mutant strains.…”