The Transcriptional Activators BAS1, BAS2, and ABF1 Bind Positive Regulatory Sites as the Critical Elements for Adenine Regulation of ADE5,7

Abstract

Adenine repression of the purine nucleotide biosynthetic genes in Saccharomyces cerevisiae involves downregulation of the activator protein BAS1 or BAS2 by an unknown mechanism. To determine the minimal cis-acting requirements for adenine regulation, hybrid promoter constructs were made between ADE5,7 promoter fragments and a CYC1-lacZ reporter. A 139-nucleotide fragment containing two BAS1 binding sites was sufficient to confer adenine regulation on the CYC1-lacZ reporter. Analysis of deletion and substitution mutations led to the conclusion that the proximal BAS1 binding site is both necessary and sufficient for regulation, whereas the distal site augments the function of the proximal site. By performing saturation mutagenesis, we found two essential regions that flank the proximal site. An ABF1 consensus sequence is within one of these regions, and mutations that impaired in vitro ABF1 binding impaired promoter activity in vivo. A second region is AT-rich and appears to bind BAS2. No substitution mutations led to high level constitutive promoter activity as would be expected from removal of an upstream repression sequence. Our results indicate that ABF1, BAS1, and BAS2 are required for ADE5,7 promoter function and that adenine repression most likely involves activator modification or a negative regulator that does not itself bind DNA.

Cooperative Pho2-Pho4 Interactions at the PHO5 Promoter Are Critical for Binding of Pho4 to UASp1 and for Efficient Transactivation by Pho4 at UASp2

“…In contrast, at the TRP4 promoter, a Pho2 binding site completely overlaps one of the two Gcn4 binding sites, and the two proteins were found to bind DNA in a mutually exclusive manner (6). The role of Pho2 (Bas2) in the activation of the ADE genes was recently investigated (21,32). Transcriptional activation of these genes requires the concerted action of Bas1 and Bas2 and is down-regulated by adenine.…”

“…Also named Bas2, Pho2 is involved in the regulation of HIS4 (3), TRP4 (6), and certain ADE genes (8). It was recently reported that at the ADE5,7 promoter, Pho2 (Bas2) binds to a site located immediately adjacent to the Bas1 site, and indirect evidence suggests that these two proteins bind DNA cooperatively (21,32).…”

Regulation of Amino Acid, Nucleotide, and Phosphate Metabolism in Saccharomyces cerevisiae

“…This hypothesis was correct, and Bas1 and Bas2 were found to be required for expression of several ADE genes in vivo and to bind to their promoter in vitro (Daignan-Fornier and Fink 1992). On the basis of proteome, transcriptome, and geneby-gene analysis, it was found that Bas1 and Bas2 activate the expression of all 10 AMP-biosynthesis genes except ADE16 (Daignan-Fornier and Fink 1992; ) and bind to a specific promoter region of these genes (Daignan-Fornier and Fink 1992; Rolfes et al 1997;Pinson et al 1998). Furthermore, Bas1/Bas2 also mediate adeninerepressible transcriptional activation of genes of other pathways metabolically connected to the purine pathway, such as histidine (HIS1, HIS4, and HIS7), glutamine (GLN1), or one-carbon-unit (SHM2, MTD1) metabolism genes ( Figure 15) (Arndt et al 1987;Springer et al 1996;.…”

“…All the genes activated by Bas1 and Bas2/Pho2 also respond to extracellular adenine, their expression being low when adenine is abundant in the growth medium (Tice-Baldwin et al 1989;Daignan-Fornier and Fink 1992;Springer et al 1996;Denis and DaignanFornier 1998). To get an insight into the molecular mechanisms linking adenine availability to transcriptional activation, Rolfes et al (1997) used chimeras between LexA and either Bas1 or Pho2. They found that lexA-Pho2 could activate transcription independently of Bas1 and in an adenine-independent way, while lexA-Bas1 activation was both Pho2 dependent and adenine responsive .…”

The Sir1 protein's association with a silenced chromosome domain

“…The HMR-E silencer also contains one binding site each for the Rap1p and Abf1p proteins, two abundant nuclear proteins with additional roles in telomere structure, transcription activation, and DNA replication and repair at other positions in the genome (Marahrens and Stillman 1992;Shore 1994;Kang et al 1995;GailusDurner et al 1996;Rolfes et al 1997;Reed et al 1999). Like ORC, both Rap1p and Abf1p must bind their sites within HMR-E to function in silencing.…”