Expression of native Ty1 elements in haploid cells. (A) β-Galactosidase activities of the 31 TY1A-lacZ fusions expressed in FYBL1-23D derivatives. Open bars, Ty1 elements defined in the text as weakly expressed elements; solid bars, Ty1 elements defined in the text as highly expressed elements; t, single-nucleotide change in the Tec1 binding site; *, β-galactosidase obtained with several strains containing an independently obtained fusion at the same Ty1 locus. Strains were grown in HC medium lacking uracil. β-Galactosidase activities were assayed as described in Materials and Methods and are shown at the right of each bar. (B) Comparison of the sum of the β-galactosidase activities obtained with the 31 TY1A-lacZ fusions (ΣTy1) with the β-galactosidase activities in the presence of galactose of Gp-PR1 and Gp-DR3, two TY1A-lacZ fusions in which the U3 region of the 5′LTR has been replaced by the GAL1 promoter at Ty1(PR1) and Ty1(DR3), respectively (34). In the presence of glucose, these two fusions gave approximately 1 U of β-galactosidase activity. (C) Northern blot analysis of total RNA in wild-type cells. For each strain, 10 μg of RNA was loaded onto the gel. Steady-state levels of TY1A-lacZ mRNA were normalized to ACT1 mRNA levels. The ratios given are relative to the TY1A(DR3)-lacZ/ACT1 ratio, arbitrarily set at 1.

Ty1 transcription is repressed by chromatin. (A) β-Galactosidase activities determined in HTA1-HTB1 and hta1-htb1Δ cells. Ty1 elements expressed at low levels and Ty1 elements expressed at high levels are defined in the text. (B) Northern blot analysis of total RNA extracted from wild-type (HTA1-HTB1), hta1-htb1Δ, ste12Δ, hta1-htb1Δ ste12Δ, and hta1-htb1Δ tec1Δ cells. For each strain, 10 μg of RNA was loaded onto the gel. Ty1 mRNA levels were normalized to ACT1 mRNA levels. Ratios given are relative to the ratio measured in the HTA1-HTB1 strain, arbitrarily set at 1. (C) β-Galactosidase activities determined in SNF2 and snf2Δ cells. (D) β-Galactosidase activities determined in wild type (HTA1-HTB1), ste12Δ, hta1-htb1Δ, and hta1-htb1Δ ste12Δ cells. A DPS1-lacZ fusion integrated at the DPS1 gene in LV261, which encodes an aspartyl-tRNA synthetase and whose expression is not regulated by chromatin, served as a control in our experiments. All strains are FYBL1-23D derivatives and were grown in HC medium lacking uracil. β-Galactosidase activities were assayed as described in Materials and Methods.

Potential Gcn4 binding sites in the 5′ LTRs of Ty1 elements. (A) Locations of the five potential Gcn4 binding sites in the 5′ LTR. A, B, C, D, and E, Gcn4^A, Gcn4^B, Gcn4^C, Gcn4^D, and Gcn4^E sites, respectively; stippled boxes, motifs recognized by the transcriptional regulators indicated below the boxes. (B) Coordinates and sequences of the Gcn4^A, Gcn4^B, Gcn4^C, Gcn4^D, and Gcn4^E sites. Coordinates are given relative to the numbering of Ty1-H3 (5). For each binding site, the order of the coordinates represents its orientation in the Ty1 sequence. Although Gcn4 recognizes a 7-bp core sequence, the SCPD database identified sequences of 6 bp as potential Gcn4 binding sites. The base ignored by SCPD is given in boldface for each Gcn4 binding site. (C) Inventory of the five potential Gcn4 binding sites in Ty1 elements.

Gcn4 overproduction activates Ty1 transcription. (A) Northern blot analysis of total RNA with strains expressing different GCN4 alleles. Lanes: Δ, Y328 transformed with YCp50; +, Y328 transformed with p164 (GCN4 URA3 CEN); C, Y328 transformed with p238 (GCN4^c URA3 CEN), which overproduces Gcn4. Strains were grown in synthetic complete medium (HC) lacking uracil. For each sample, 10 μg of total RNA was loaded onto the gel. The HIS4 gene, which is activated by Gcn4, served as a positive control. Ty1 and HIS4 mRNA levels were normalized to those of ACT1. The mRNA ratios given are relative to the ratio obtained for the Y328/p164 strain, arbitrarily set at 1. (B) β-Galactosidase activities of TY1A-lacZ fusions in wild-type cells (GCN4) and in wild-type cells overexpressing GCN4 (GCN4^c). Ty1 “low,” weakly expressed Ty1 elements; Ty1 “high,” highly expressed Ty1 elements; GCN4, strains transformed with pRS313 (HIS3 CEN); GCN4^c, strains transformed with pAM25 (GCN4^c HIS3 CEN). Strains are FYBL1-23D derivatives. Cells were grown in HC medium lacking uracil and histidine.

Amino acid limitation affects Ty1 transcription in the absence of Gcn4. (A) Northern blot analysis of cells grown under histidine starvation. GCN4, strain Y328 transformed with p164 (GCN4 URA3 CEN); gcn4Δ, Y328 transformed with YCp50. 3AT (10 mM) and histidine (120 μM) were added (+) or not (−) to cultures grown at 22°C in minimal medium supplemented with arginine, isoleucine, tryptophan, leucine, and valine (SDc), as described previously (43). For each sample, 10 μg of total RNA was loaded onto the gel. Ty1 and HIS4 mRNA levels were normalized to those of ACT1. The mRNA ratios given are relative to that for the GCN4 strain grown in SDc lacking 3AT and histidine. The HIS4 gene, which is activated by Gcn4, served as a positive control. (B) Northern blot analysis with strains expressing different GCN4 alleles. Δ, Y328 transformed with YCp50; +, Y328 transformed with p164; C, Y328 transformed with p238 (GCN4^c URA3 CEN), which overproduces Gcn4. SDm, minimal medium supplemented with arginine and leucine. SDc is explained in the legend to panel A. In SDc, GCN4 expression is not activated, whereas GCN4 expression is activated in SDm due to amino acid limitation. Precultures and cultures were grown as described previously (43), except that cultures were grown to mid-log phase at 22°C. Ty1 and HIS4 mRNA levels were normalized to those of ACT1. The mRNA ratios given are relative to the ratio under the nonactivating conditions in SDc medium with strain Y328/p164. (C) β-Galactosidase activities of the TY1A(PR1)^h-lacZ fusion under conditions of histidine starvation. Growth conditions are described in the legend to panel A. gcn4Δ, strain LV568 transformed with pRS313 (HIS3 CEN) (44). GCN4, strain LV568 transformed with pAM19 (GCN4 HIS3 CEN). Strains are Y328 derivatives.

The Gcn4^E binding site contributes to the activation of Ty1 transcription by Gcn4. (A) β-Galactosidase activities of the different HIS4-lacZ constructs in strains grown under normal conditions or under conditions of histidine starvation. 3AT was added to the cultures as described in the legend to Fig. 5A. GCN4, strain LV555 transformed with pAM19 (GCN4 HIS3 CEN); gcn4Δ, strain LV555 transformed with pRS313; WT, HIS4-lacZ fusion; ΔUAS, HIS4^ΔUAS-lacZ fusion; 3H, HIS4^3H-lacZ fusion; 3L, HIS4^3L-lacZ fusion. The different HIS4-lacZ constructs are carried on 2μm URA3 plasmids as described in Materials and Methods. LV555 is a Y328 derivative. (B) Gel retardation assays. 3H, DNA probe containing 3 copies of the Gcn4^E motif; 3L, DNA probe without the Gcn4^E motif. Gcn4, purified His-tagged Gcn4 protein; +c, cold competitor (250 ng of pHIS4^3H-lacZ); I, II, and III, His-tagged Gcn4/DNA complexes.

Model for transcriptional activation of Ty1 elements. Shaded ovals, nucleosomes; shaded rectangles, Ty1 5′ LTR; open rectangles, transcriptional regulatory region of Ty1; solid box, Gcn4 binding site (only one site is shown for clarity); cross-hatched boxes, Ste12 and Tec1 binding sites. (A) Ty1 elements are contained within repressive chromatin, which is not removed at the Ty1 elements expressed at low levels because they lack the Gcn4 motifs. (B) Gcn4 binds to Ty1 sequences harboring Gcn4 binding sites. (C) Gcn4 recruits Swi/Snf and SAGA. (D) Swi/Snf and SAGA displace nucleosomes, allowing binding of Ste12 and Tec1 to their sites. (E) Ste12 and Tec1 activate Ty1 transcription (wavy arrow). Events B to E occur at the highly expressed elements.