Expression of the arginase (CAR1) gene in Saccharomyces cerevisiae is induced by arginine or its analog homoarginine. Induction has been previously shown to require a negatively acting upstream repression sequence, which maintains expression of the gene at a low level in the absence of inducer. The objective of this work was to identify the cis-acting elements responsible for CAR1 transcriptional activation and response to inducer. We identified three upstream activation sequences (UASs) that support transcriptional activation in a heterologous expression vector. Two of these UAS elements function in the absence of inducer, whereas the third functions only when inducer is present. One of the inducer-independent UAS elements exhibits significant homology to the Sp1 factor-binding sites identified in simian virus 40 and various mammalian genes.

DNA fragments previously shown to be required for expression of the CAR1 (arginase) gene in Saccharomyces cerevisiae and to support transcriptional activation of a reporter gene in a heterologous expression vector were shown to bind purified regulatory protein ABF-1. Two ABF-1 sites were identified in the CAR1 upstream region, one to which ABF-1 protein bound with high affinity and a second to which it bound much less avidly. The higher-affinity ABF-1 binding site upstream of CAR1 was an effective competitor of the HMRE, ARS1 B domain, and COR2-GFI binding sequences for protein binding. Point mutations in the CAR1 high-affinity ABF-1 binding site resulted in a 12-fold loss of transcriptional activation of a reporter gene compared with the wild-type CAR1 DNA fragment. These data are consistent with the suggestion that ABF-1 protein is one of the transcription factors involved in expression of the CAR1 gene.

Regulated expression of the inducible arginase (CARl) gene of Saccharomyces cerevisiae has been shown to require (2, 6, 19-21, 27b). However, it was recently shown that only arginine transport is NCR sensitive; the apparent NCR sensitivity of CARl expression is in reality NCR-sensitive inducer exclusion (7).The CAR1 upstream regulatory region contains four discrete cis-acting elements: two inducer-independent upstream activation sequences (UASs), UASCl and UASC2; an inducer-dependent UAS, UAS,; and an upstream repression sequence, URS1 (11,13,15,23,[28][29][30][31]. The URS1 site is found upstream of many yeast genes and has been shown to bind a heteromeric protein (14,15,30). The functional integration of these elements and the proteins associated with them is proposed to mediate controlled CARl expression (30). In the absence of inducer, UASC1 and UASc and their associated proteins are capable of activating transcription but are prevented from doing so by the strong negative action of URS1 and its associated proteins. When arginine is added, UASJ becomes functional, the combined action of all three UAS elements operating simultaneously overcomes the negative action of URS1, and expression occurs (13).For an understanding of the biochemical mechanisms through which controlled expression is accomplished, it is necessary to identify all the proteins that interact with the CAR] cis-acting elements. We recently reported that UASC1 and UASC2 contained ABF-1 binding sites (11). Both sites were required for the support of wild-type levels of transcriptional activation. However, one of the sites was significantly more important than the other.Work from several other laboratories suggested that the ABF-1 transcription factor often functions in association with a second factor, RAP1 (3-5, 16, 27 303 (25). The RAP1 DNA binding domain is located between residues 361 and 596 of the 827-amino-acid protein (9). Therefore, the truncated protein retains the DNA binding site. Extracts from this strain produce a higher-mobility band shift of HIS4 upstream sequences than extracts from the wild-type strain do (25).The Escherichia coli strain used for cloning was HB101 (hsd-20 levB supE44 ara-14 galK2 lacY] proA2 rpsL20xyl-5 mtl-1 recA13 mcrB). Yeast

Induced production of arginase (CAR1) enzyme activity and steady-state CAR1 mRNA in Saccharomyces cerevisiae requires wild-type ARG80/ARGRI and ARG81/ARGRII gene products. We demonstrate here that these gene products, along with that of the MCM1 gene, are required for the inducer-dependent USAI-A, UASI-B and UASI-C elements to function but they are not required for operation of inducer-independent CAR1 UASC1 or UASC2. Through the use of single and multiple point mutations, the CAR1 UASI-B and UASI-C elements were demonstrated to be at least 23 bp in length. Moreover, simultaneous mutation of both ends of an elements gave stronger phenotypes than mutations at either end. The center of the element was more sensitive to mutation than were the ends.

Expression of the Saccharomyces cerevisiae arginase (CAR1) gene is regulated by induction and nitrogen catabolite repression (NCR). Arginine was demonstrated to be the native inducer. CAR1 sensitivity to NCR has long been accepted to be accomplished through a negative control mechanism, and cis-acting sites for it have been hypothesized. In search of this negatively acting site, we discovered that CAR1 sensitivity to NCR derives from regulated inducer (arginine) exclusion. The route of catabolic entry of arginine into the cell, the general amino acid permease (GAP1), is sensitive to NCR. However, CAR1 expression in the presence of sufficient intracellular arginine is NCR insensitive.