A single Saccharomyces cerevisiae upstream activation site (UAS1) has two distinct regions essential for its activity.

Abstract

Several site-directed mutagenesis regimens were used to generate single-and multiple-base substitutions in the upstream activation site UAS1 of the Saccharomyces cerevisiae CYCI gene. Mutations resulting in large reductions in activity of the site lie in two distinct regions. Six single-base changes in a region A, between -288 and -285, all resulted in a 15-fold reduction in activity. Synthetic sites built up solely of multimers of the -289 to -285 sequence ACCGA behaved as carbon catabolite-sensitive UASs. In addition, substitution mutations in a second region, at nucleotides -266 and -265, virtually eliminated UAS1 activity. These mutations abolished the binding of a heme-dependent protein factor in vitro. Thus, UAS1 contains two essential regions both of which are required for its activity.Upstream activation sites (UASs) are cis-acting elements found upstream of TATA boxes that are required for efficient transcription of all Saccharomyces cerevisiae genes analyzed to date (for a review, see reference 10). UASs constitute a primary route by which gene-specific regulation is conferred (5, 12-14, 21, 26).The sites appear to have several features in common with analogous sites, termed enhancers, identified in higher eucaryotes (reviewed in references 4, 9, 19). For example, the yeast sites function at variable distances from the TATA box or when their orientation with respect to the TATA box is inverted (11). The activity of UASs and enhancers very likely depends on trans-acting proteins that bind to the sites. In mammalian systems both the mouse mammary tumor virus (MTV) enhancer and the simian virus 40 distal regulatory region consist of composites of short repeated elements. The MTV element is an octamer to which the glucocorticoid receptor binds (28), and the simian virus 40 element is a hexamer to which Spl binds (7). In yeasts, galactose inducibility can be mediated by a 17-base-pair (bp) dyad to which the GAL4 protein binds (3,8). Further, a hexamer has been identified genetically as the UAS mediating general control of many amino acid biosynthetic genes (5, 15, 16). Binding of the product of the GCN4 locus to this sequence will occur in vitro but is dependent on sequences that flank the hexamer (18).Regulation of the CYCI gene which encodes iso-1-cytochrome c is governed by two distinct UAS sites, UAS1 located at -275 and UAS2 located at -225 ( Fig. 1) (2). By fusing these sites separately to the LEU2 TATA box, we showed that their activity requires heme: both sites display a very low level of activity under heme-deficient conditions and are induced about 200-fold by the addition of heme to growth media (12, 13). Under heme-sufficient conditions, the sites are further derepressed by shifting from glucose medium to one containing a nonfermentable carbon source, e.g., lactate. UAS1 may also be derepressed by the addition of the heme analog deuteroporphyrin IX to glucose medium. Each UAS site can function independently of the other, and the effect of both sites in the intact promoter is roughly additive. Al...

Iron Regulation through the Back Door: Iron-Dependent Metabolite Levels Contribute to Transcriptional Adaptation to Iron Deprivation in Saccharomyces cerevisiae

“…Thus, the effect of Hap4 on the iron responsiveness of CYC1 is transmitted via both activating domain UAS2 and, to a lesser extent, UAS1. The fact that this Hap4-dependent modulation was mediated via both upstream activating domains is in accordance with previous findings (28). Taken together, these data demonstrate that the transcriptional activators Hap1 and Hap4 play decisive roles for the iron-responsive expression of CYC1.…”

A regulatory region responsible for proline-specific induction of the yeast PUT2 gene is adjacent to its TATA box.

“…The HIS4 and CUP) genes require two copies of the UAS for wild-type levels of gene products (28,60). Genes with more than one type of UAS element have also been described (20,35,39). The HIS4 gene resembles PUT?…”

Nucleo‐mitochondrial interactions in yeast mitochondrial biogenesis

“…As pointed out above, a number of other proteins contribute to transcriptional activation of UAS2 and the action of these remains to be investigated. In UASI, mutational analysis has also defined two domains, both of which are required for binding of HAP1 and for activity [131]. The first of these, region A, binds an as yet poorly defined protein called RAF (region A factor [112]) and methylation-interference footprint analysis suggests that protein -protein interactions between the two factors may contribute to the formation of a stable complex.…”