A cis-acting element present in multiple genes serves as a repressor protein binding site for the yeast CAR1 gene.

Abstract

Induction of the arginase (CAR1) gene expression in Saccharomyces cerevisiae has previously been shown to require participation of a cis-dominantly regulated upstream repression sequence (URS). Deletion of this element results in high-level expression of the CAR1 gene without inducer. To determine the structure of the CAR1 URS element, we performed a saturation mutagenesis. Results of the mutagenic analysis indicated that the CAR1 URS was a 9-base-pair palindromic sequence, 5'-AGCCGCCGA-3'. A DNA fragment containing this sequence was shown to bind one or more proteins by a gel shift assay. DNA fragments containing point mutations that completely eliminated URS function were not effective competitors in this assay, whereas those which supported URS function were effective competitors. Sequences in the 5'-flanking regions of 14 other genes were found to be homologous to the CAR1 URS. These sequences were shown to support varying degrees of URS function in the expression vector assay, to bind protein as demonstrated by the gel shift assay, and to compete with a DNA fragment containing the CAR1 URS for protein binding. These results indicate that the CAR1 URS element possesses the characteristics of a repressor binding site. Further, they are consistent with the suggestion that sites homologous to the CAR1 URS may be situated in the 5'-flanking regions of multiple unrelated yeast genes. The widespread occurrence of this element raises the possibility that it is the target site for one or more negatively acting general transcription factors.

Analysis of a Meiosis-Specific URS1 Site: Sequence Requirements and Involvement of Replication Protein A

“…This site, which we will refer to as URS1 C , functions to repress CAR1 expression in the absence of arginine and also works as a strong repressor element in heterologous promoters. URS1 C consists of a 9-bp palindromic sequence (5Ј-TCGGCGGCT-3Ј); there is an 8-of-9-bp identity between the HOP1 and CAR1 URS sites within this core region, suggesting that the same proteins may function at both of these sites (26,43,46). This prediction is supported by the observation that the CAR1, SPO13, and IME2 URS1 sites require the presence of Ume6, also known as Car80, to repress transcription (6,34,42).…”

“…Analyses of the URS1 sites from the meiosis-specific genes SPO13, HOP1, and IME2 have shown that they function as repressor sites during mitotic growth and either are derepressed or serve as activator sites during meiosis (6,10,46). The canonical and perhaps most extensively studied URS1 site is part of the promoter of the nonmeiotic gene CAR1, a gene which codes for arginase and is required for nitrogen metabolism (26,43). This site, which we will refer to as URS1 C , functions to repress CAR1 expression in the absence of arginine and also works as a strong repressor element in heterologous promoters.…”

Characterization of a transcriptional control element involved in proliferation of peroxisomes in yeast in response to oleate

“…Upstream of the putative ORE at least two factors have been shown to bind (Einerhand et al, 1991 ;Einerhand et al, 1992;Einerhand et al, unpublished observations). These include the abundant yeast transcription factor ABFl and a factor interacting with a sequence motif resembling the repressor-binding site in the 5' flanking region of the arginase gene ( C A M ) , identified by Cooper and colleagues (Sumrada and Cooper, 1987;Luche et al, 1990). Furthermore, it was demonstrated that the transcription factor ADRl is required for derepression of the FOX3 gene (Simon et al, 1991).…”

Functional Domain Mapping and Subcellular Distribution of Dal82p in Saccharomyces cerevisiae

“…HA-tagged antibody with horseradish peroxidase-conjugated secondary antibody (goat anti-mouse IgG; Bio-Rad) were used. EMSAs were performed using 10 g of calf thymus ϩ 50 ng of radiolabeled DNA fragment and 1-5 g of protein (crude cell extract) (49). Urea amidolyase activity was determined in nystatin-permeabilized cells/transformants (4,50,51).…”