Upstream activation sites of the CYCI

The ability of the upstream activation sites (UASs) of the yeast CYCI gene to function when inverted or when positioned downstream of the "TATA box" is investigated. Inversion of a 130-base-pair DNA fragment bearing the UASs leaves the activity of the sites almost completely intact. In contrast, positioning the sites downstream of the TATA box or in the intron of a CYCI-ribosomal protein 51-iacZ tribrid gene almost totally abolishes their activity. In the latter construct, the separation between the UASs and TATA box is roughly equivalent to that between the elements in the intact CYCI promoter region. The UASs are shown not to interrupt transcription or splicing in this construct since a GALIO UAS positioned upstream of the TATA box gives rise to galactoseinducible expression of the tribrid gene. The inability of the UASs to function in the intron is partly due to sequences between the intron and the TATA box that block the activation signal. However, a large component of the inactivity of the sites in the intron appears to be their downstream location. This result is discussed in light of possible mechanisms of upstream activation in yeast. In higher eukaryotes, RNA polymerase II typically initiates transcription 30-40 base pairs (bp) downstream of the canonical "TATA box" sequence (1, 2). The frequency of initiation, for many genes, is determined by sequences lying upstream of the TATA box, one class of which is found <100 bp away and regulates transcription in response to particular physiological signals (3-6). A second class of upstream sequences, called enhancers, lies in a more remote position and can mediate hormonal responsiveness or cell type-specific regulation in mammals (2, 7-11, 26). Several features of cloned enhancers are revealed when the sites are manipulated by recombination in vitro. For example, the sites can function when transposed to a location several kilobases away from the TATA box or when their orientation with respect to the transcriptional unit has been inverted. Although many enhancers naturally occur upstream of the TATA box, some are found to the 3' side of the affected transcriptional unit (12-14). Many enhancers that are naturally found in an upstream location will function when positioned 3' to the transcriptional unit (10, 15, 16). In yeast, TATA boxes have been shown to be functionally important in several cases (17, 18). Sequences that lie upstream of the TATA box that activate transcription in response to particular physiological signals appear to occur frequently. Such upstream activation sites (UASs) have been found for genes involved in histidine biosynthesis, HIS3 (17) and HIS4 (19), a gene involved in galactose catabolism, GALIO (20), the CYCI gene, encoding the iso-1-cytochrome c (18, 21), and a gene involved in leucine biosynthesis, LEU2 (22). These sites lie between -30 and -300 (from the transcriptional initiation site) and activate transcription in response to particular physiological signals (for review, see ref. 23). Genetic studies suggest that UASs are regulated by proteins encoded by specific regulatory loci (21). The CYCJ gene contains tandem sites, designated UAS1 and UAS2, which lie at about -275 and -225, respectively (21). These sites are differentially regulated as follows: both UASs are uninduced when cells are grown under conditions of deficiency of heme, the cytochrome cofactor; UAS1 is partly induced in cells grown under heme sufficiency in media with glucose as carbon source; both UAS1 and UAS2 are fully and equally active in cells grown under derepressing conditions in media containing a non-fermentable carbon source. In several cases hybrid promoters have been constructed consisting of the UAS of one gene and the TATA box of another (20, 21). These hybrids direct transcription initiation at the correct site(s) specified by the TATA box region and are regulated in a manner determined by the UAS in cis. These and other experiments suggest that there is some flexibility in the spacing between UASs and TATA boxes. In this report, it is determined whether the CYCJ UASs function when inverted with respect to the TATA box and when placed to the downstream side of that element. To aid in the design of the latter experiment, the effects of distance separating the UASs from the TATA box were also assessed. The results obtained strengthen the credibility of one possible molecular mechanism of UAS activation and weaken the credibility of another. MATERIALS AND METHODS Strains and Plasmids. Yeast strains BWG2-9A-1 (Mata, his4-519, ade6, ura3-52) and BWG1-7a (Mata, leu2-2,2-112, his4-519, adel-100, ura3-52), TM1 (hemi, ura3-52), and LG1-2D (MATa, trpl, ura3-52, gal80) (21) were employed for all assays described. All plasmid constructs are derived from pLGA-312S (18). Assays and Media. f3-Galactosidase assays were performed as described (24). Units are according to Miller (25). Cells were grown in yeast nitrogen base without amino acids for 1galactosidase assays or RNA preparations. DNA Manipulations. All methods including plasmid construction, DNA or RNA isolation, and S1 mapping were as described (18, 24). Insertions. pLGA-312S was digested with Sal I and Escherichia coli DNA that had been cut with Xho I was inserted. The Sal I and Xho I ends are complementary. Insert sizes were determined by restriction analysis. UAS inversion. pLGA-312 was digested with Sma I and Xho I, the Xho I end rendered flush with DNA polymerase, Abbreviations: UAS, upstream activation site; bp, base pair(s); kb, kilobase(s); El and E2, exons 1 and 2, respectively. 7860 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. NatL Acad. Sci. USA 81 (1984) 7861