Participation of RAP1 protein in expression of the Saccharomyces cerevisiae arginase (CAR1) gene

Abstract

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

Combinatorial Regulation of theSaccharomyces cerevisiae CAR1(Arginase) Promoter in Response to Multiple Environmental Signals†

“…The arbitrarily defined UAS CI and UAS CII regions supported constitutive reporter gene expression when cloned into a heterologous expression vector (43,44,46). Genetic and in vitro biochemical studies demonstrated the UAS C regions potentially to contain two Abf1p-binding sites (44) and five Rap1p-binding sites (46).…”

“…The arbitrarily defined UAS CI and UAS CII regions supported constitutive reporter gene expression when cloned into a heterologous expression vector (43,44,46). Genetic and in vitro biochemical studies demonstrated the UAS C regions potentially to contain two Abf1p-binding sites (44) and five Rap1p-binding sites (46). However, the degrees to which individual sites were required for the DNA fragments containing them to support high-level ␤-galactosidase production when cloned into a CYC1-lacZ expression vector varied (44,46).…”

Tripartite structure of the Saccharomyces cerevisiae arginase (CAR1) gene inducer-responsive upstream activation sequence

“…In addition to the three 27-bp UAS1 elements described in this article, a further nine specific protein-binding sites have been previously reported to be present in the 5'-flanking region of CARI (Fig. 11) (12,13). They facilitate relation of the earlier work to that described in this article.…”

“…According to this model, the negative action of the strong URSI element balances the positive action of the strong, constitutively functioning UASCl and UASc2 elements (11)(12)(13)(14)(15). The balance is then tipped in the direction of expression when arginine is present, or quiescence when it is not, by operation of the inducer-dependent UASJ element (11)(12)(13)(14)(15).…”

“…The balance is then tipped in the direction of expression when arginine is present, or quiescence when it is not, by operation of the inducer-dependent UASJ element (11)(12)(13)(14)(15).…”