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

Abstract

Arginase (CARI) gene expression in Saccharomyces cerevisiae is induced by arginine. The 5' regulatory region of CAR) contains four separable regulatory elements-two inducer-independent upstream activation sequences (UASs) (UASCl and UASC2), an inducer-dependent UAS (UASJ), and an upstream repression sequence (URSI) which negatively regulates CA4R and many other yeast genes. Here we demonstrate that three homologous DNA sequences originally reported to be present in the inducer-responsive UASI are in fact three exchangeable elements (UASI.A, UASI-B, and UASI.C). Although two of these elements, either the same or different ones, are required for transcriptional activation to occur, all three are required for maximal levels of induction. The elements operate in all orientations relative to one another and to the TATA sequence. All three UAS, elements bind protein(s); protein binding does not require arginine or overproduction of any of the putative arginine pathway regulatory proteins. The UASI-protein complex was also observed even when extracts were derived from arg80/argRI or arg81/argRll deletion mutants. Similar sequences situated upstream ofARG5,6 andARG3 and reported to negatively regulate their expression are able to functionally substitute for the C4RI UASJ elements and mediate reporter gene expression. CARI (arginase) gene expression in Saccharomyces cere-visiae has been studied as a model of biochemical mechanisms underlying eucaryotic transcription and the cis-and trans-acting factors by which it is regulated. CARI mRNA production is induced to a high level when arginine, the native inducer, is added to the culture medium (1, 5, 17-20, 27, 36). High-level CARI mRNA production is not observed, however, when a readily catabolized nitrogen source such as asparagine or glutamine is present (1,3,5,(18)(19)(20)27); i.e., CARI expression exhibits apparent sensitivity to nitrogen catabolite repression (NCR) (1,3,5,(18)(19)(20)27). CARI NCR sensitivity was generally accepted to result from a discrete transcriptional control process. However, it has recently been shown that CARI sensitivity to NCR occurs via NCRmediated inducer exclusion (4).Dissection of the CAR1 5'-flanking region to identify the sequences required for transcriptional activation and its regulation revealed four separate elements (11-13, 15, 22, 28-31). The first element identified was URS1, a repressorbinding site that functions not only in CARI but also in many other genes, e.g., those participating in meiosis, mating type control, heat shock response, carbon metabolism, inositol metabolism, and oxidative metabolism (2, 9, 10, 14, 15, 22, 28-31, 35, 37). The protein that binds to this site has been purified, and the genes encoding it have been cloned and sequenced (14, 14a). The CARI promoter also contains three discrete upstream activation sequences (UASs) (11-13, 28, 31, 33 currently under study. Thus far, three RAP1 sites and one ABF1 site have been identified (12, 13). However, additional evidence suggests that one or more sites rem...

Nitrogen Starvation and TorC1 Inhibition Differentially Affect Nuclear Localization of the Gln3 and Gat1 Transcription Factors Through the Rare Glutamine tRNACUG in Saccharomyces cerevisiae

“…The high levels of CAR1 mRNA likely derived from two sources. First, the CAR1 promoter contains multiple demonstrably functional cis-acting elements (12 sequences), including three CAR1 UAS I elements that mediate strong arginineinduced CAR1 transcription and one that is homologous to a Gln3/Gat1 binding site (Kovari et al 1990(Kovari et al , 1993aViljoen et al 1992). The vacuole contains millimolar levels of arginine that are mobilized during starvation; this induces arginase (CAR1) production, accounting for the high levels of CAR1 expression observed (Wiemken et al 1970;Wiemken and Durr 1974;Zacharski and Cooper 1978;Sumrada and Cooper 1978).…”

The E2 Ubiquitin Conjugase Rad6 Is Required for the ArgR/Mcm1 Repression ofARG1Transcription

“…Arginine repression requires a DNA binding complex of ArgR proteins, Arg80/ArgRI and Arg81/ArgRII, as well as Mcm1 (1,8,[23][24][25][26]51,52,57,58). Interestingly, this same ArgR/Mcm1 complex is required for induction of CAR1 and CAR2, which are required for arginine catabolism (43,52,67). For both activation and repression, the ArgR/Mcm1 complex binds to upstream arginine control (ARC) elements (10,17,21,24,51).…”

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

“…The ability of the three UAS I elements to support inducer-dependent reporter gene expression varied, with the 5Ј-and 3Ј-most elements being the least and most effective, respectively (81). Although at least two of the CAR1 UAS I elements are required for induced transcription, they are not required to be situated in a specific orientation relative to one another or to the TATA element (45,81). Moreover, a single UAS I element derived from ARG5,6 is similarly capable of transcription, further demonstrating the functional independence of the elements (45).…”

“…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). The UAS I region was shown to consist of three tandem sequences required for arginine-inducible reporter gene expression (43,45,81). The ability of the three UAS I elements to support inducer-dependent reporter gene expression varied, with the 5Ј-and 3Ј-most elements being the least and most effective, respectively (81).…”

“…The UAS I region was shown to consist of three tandem sequences required for arginine-inducible reporter gene expression (43,45,81). The ability of the three UAS I elements to support inducer-dependent reporter gene expression varied, with the 5Ј-and 3Ј-most elements being the least and most effective, respectively (81). Although at least two of the CAR1 UAS I elements are required for induced transcription, they are not required to be situated in a specific orientation relative to one another or to the TATA element (45,81).…”