Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press GALI-GALIO divergent promoter region of Saccharomyces cerevisiae contains negative control elements in addition to functionally separate and possibly overlapping upstream activating sequences Robert W. West Jr., Shiming Chen, Henry Putz, Geraldine Butler, 1 and Mary Banerjee Department of Biochemistry and Molecular Biology, SUNY Health Science Center, Syracuse, New York 13210 USA The upstream activating sequence (UASG) of the adjacent and divergently transcribed GALl and GALIO promoters of Saccharomyces cerevisiae regulates the induction of the corresponding genes in response to the presence of galactose. We constructed chimeric yeast promoters in which a different UAS, UASc from the iso-l-cytochrome c (CYC1) gene of S. cerevisiae, was fused at different locations upstream of GALl (UASc-- GALl promoters) or GALIO (UASc- GALIO promoters) and used to monitor the activity of UASG in cells grown in the presence or absence of galactose. Though the CYC1 promoter is fully induced in yeast grown in glycerol medium, UASc-GAL chimeric promoters containing UASG were repressed as much as 400-fold (UASc-GAL1) or 1350-fold (UASc- GALIO) in this growth medium. Several distinct portions of the GAL1- GALIO divergent promoter region blocked the UASc-induced expression of the GALl and GALIO promoters, whereas others did not, suggesting that several distinct negative control elements are present that may repress transcription of GALl and GALIO in the absence of galactose. The approximate locations of these negative control elements were delimited to sites adjacent to or possibly overlapping the sites at which the positive control protein GAL4 binds in UASG. Deletion derivatives of GAL4 that fail to induce transcription from the wild-type GAL promoters but retain the DNA binding domain significantly derepressed the expression of the UASc-GAL chimeric promoters. These results, combined with those of earlier studies, suggest the possibility that GAL4 normally induces transcription of GALl and GALIO by blocking the activity of these negative control elements, in addition to stimulating transcription by a mechanism of positive control. [Key Words: Upstream activating sequence; negative control; GALl- GALIO divergent promoter; Saccharomyces cerevisiae; CYC1 gene] Received June 9, 1987; revised version accepted October 6, 1987. Transcriptional control of eukaryotic protein-coding distal regulatory elements have been termed upstream genes requires specific regulatory sequences located ad- activating sequences, or UASs (Guarente 1984). jacent to each gene. Proximal regulatory sequences in- One well-characterized yeast UAS is UASG, which clude the TATA box, which in yeast is known to be in- controls the adjacent and divergently transcribed GALl volved in determining the precise start sites for tran- and GALI O genes (Guarente et al. 1982; Johnston and scription initiation (Chen and Struhl 1985; Hahn et al. Davis 1984; West et al. 1984; Yocum et al. 1984). UASG 1985; Nagawa and Fink 1985; McNeil and Smith 1986). is about 120 bp in size (nomenclature of Giniger et al. Distal regulatory sequences (upstream promoter ele- 1985), resides about midway between the translation ments) respond to specific physiological stimuli to con- start sites of GALl and GALIO, and is required for galac- trol the amount of transcription initiating downstream tose-mediated induction of both genes. In galactose (Gal) (Guarente et al. 1984; Giniger et al. 1985; Hope and medium, the positive control protein GAL4 binds to Struhl 1985; Arndt and Fink 1986; McKnight and Tjian four related, dyad-symmetric sequences in UASG and in- 1986; Pfeifer et al. 1987). In Saccaromyces cerevisiae the duces transcription of GALl and GALI O (Brain and Kornberg 1985; Giniger et al. 1985). In glycerol (Gly)me- dium, though GAL4 is produced constitutively (John- ~Permanent address: Department of Genetics, Trinity College, Dublin, Ireland. ston and Hopper 1982; Laughon and Gesteland 1982), its 1118 GENES& DEVELOPMENT 1:1118-1131 © 1987 by Cold Spring Harbor Laboratory ISSN 0890-9369/87 $1.00 Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Negative elements in a yeast GAL promoter activity is inhibited by the negative regulatory protein elements in the GAL1-GALIO divergent promoter re- GAL80 and transcription of GALl and GALI O is pre- gion. To pursue this, we fused a 150-bp fragment con- vented (Lue et al. 1987). GAL80 probably inhibits GAL4 taining UASc from the iso-l-cytochrome c (CYC1) gene by binding to a distinct region at its carboxyl terminus of S. cerevisiae (Guarente et al. 1984; Pfeifer et al. 1987), and blocking a specific domain that is rich in acidic at the end points of 5' deletions of GALl or GALl 0, as amino acids (see Struhl 1987) and is involved in GAL4's shown in Figure 1. Our rationale was that UASc would positive control (transcription-activating) function provide the GAL promoters a basal level of expression (Johnston et al. 1987; Ma and Ptashne 1987b). independent of galactose and GAL4, allowing us to The DNA-binding domain of GAL4 is located within monitor the capacity of portions of UASG and flanking the amino-terminal 75 amino acids of the 881-amino- sequences to inhibit UASc-induced expression of GALl acid protein (Brent and Ptashne 1985; Keegan et al. 1986; or GALI O. Activation of CYC1 transcription by UASo Johnston 1987; Johnston and Dover 1987). Data derived though inhibited about fivefold by glucose, is essentially from in vivo DMS protection (Giniger et al. 1985), constitutive under the growth conditions we normally DNase I footprinting (Lohr and Hopper 1985), and pho- use to induce or repress the GAL promoters (galactose tofootprinting studies (Selleck and Majors 1987a, b) indi- vs. glycerol and lactate medium, respectively). cate that GAL4 may be bound at UASG in Gly medium To measure the amount of expression from the hybrid as well as Gal medium. This result, and the fact that promoters, the GALl and GALI O genes were fused to GAL80 represses transcription of GALl and GALI O by the Escherichia coli lacZ gene, and levels of transcrip- blocking GAL4's transcription-activating domain, sug- tion were determined by assaying for fl-galactosidase gests a model where galactose induces transcription by produced in yeast. The UASc-GAL-lacZ gene fusions causing GAL80 to dissociate from GAL4 molecules were maintained on multicopy plasmids derived from bound at UASG, thus exposing GAL4's transcription-ac- YEp24, as described previously (West et al. 1984; Yocum tivating domain to the cellular transcription apparatus et al. 1984). Since UASc and UASG are differentially reg- (Johnston et al. 1987; Ma and Ptashne 1987b; Selleck ulated, expression of the hybrid UASc-GAL promoters and Majors 1987b). should reflect the physiological and genetic conditions Four other S. cerevisiae genes, GAL7, GAL2, GAL80, normally controlling CYC1 or GAL gene transcription. and MEL1, are also induced by GAL4 and have GAL4 For this purpose, the plasmids were transformed into the binding sites in their 5' control regions (Bram et al. yeast strains YM256 (GAL4 +) or YM335 (Agal4)and the 1986). The MEL1 and GAL80 genes are transcribed at a transformants grown in synthetic medium containing detectable level in uninduced cells (Post-Beittenmiller et either glycerol and lactate (Gly)or galactose plus glyc- al. 1984; Shimada and Fukasawa 1985), whereas GALl, erol and lactate (Gal), prior to assaying for f3-galactosi- GALIO, GAL7, and GAL2 are not (St. John and Davis dase production. 1981; West et al. 1984; Yocum et al. 1984; Tajima et al. 1986; Tschopp et al. 1986). Previous deletion-mapping Expression in GAL medium analysis of the 600-bp GALl-GALl 0 divergent promoter region revealed that a certain portion of UASG (located The activities of the hybrid promoters shown in Figure proximal to the GALl promoter), when deleted, in- 1, when transformed into YM256 (GAL4 ÷) and grown in creased the uninduced level of GALl transcription from Gal medium, are presented in Table 1. Only hybrid pro- an undetectable level to about 5% of the fully induced moters containing a single copy of UASo inserted in the level (West et al. 1984). This raised the possibility that a normal orientation with respect to a TATA box, are in- negative control element(s)is also present in UASG that cluded in Table 1 (see Materials and methods). The re- normally represses transcription of GALl and GALl 0 in sults indicated that if UASG was present in a particular uninduced cells. UASc-GAL1 or UASc-GAL10 hybrid promoter, expres- Here we show evidence suggesting that multiple nega- sion of the UASc-GAL1- or UASc-GALIO-lacZ fu- tive control elements are present in the GAL1-GALIO sions was normally induced, and f3-galactosidase levels divergent promoter region, which may account, in part, were often as much as twofold higher than in cells con- for the lack of detectable expression of the respective taining a respective GALl- or GALI O--lacZ fusion genes in Gly medium. The negative control elements lie lacking UASc. If UASG was absent in a given chimeric adjacent or possibly overlap the GAL4 binding sites in promoter, as with plasmids UASc-GAL1-8 and UASc- UASG, and neither GAL4 nor GAL80 is required for their GALl-9 in Table 1 for example, expression derived function. Deletion derivatives of GAL4 that apparently solely from the activity of UASc. These results show bind to UASG, but fail to activate transcription of the that UASc does not affect the activity of UASG other wild-type GAL genes, significantly block the activity of than to increase the total amount of expression from the the negative control elements, suggesting that normally hybrid promoters and that the GALl and GALI O pro- GAL4 regulates the activity of this repression mecha- moters can be induced by UASc alone when UASG is nism.