Transcription of RP genes decreased under amino-acid starvation in a Gcn4p-dependent manner. (A) Transcription of RP genes under amino-acid starvation. JS143-7D strain growing at early exponential phase was re-cultivated with 3-AT (20 mM) or with limited amino acids (10% of the concentrations originally required) or with sulfometuron methyl (SM, 10 μg/ml). Each transcript level was examined by northern blot with specific probes. The mRNA levels were measured and normalized to the level of ACT1. Experiments were performed in triplicate with three independent colonies. (B) Gcn4p-dependent regulation of RP transcription under amino-acid starvation. Exponentially growing cells of both GCN4 wild-type (JS143-7D, GCN4) and deletion strain (YJK101, Δgcn4) in SC—his were treated with 3-AT (80 mM) or an equal volume of distilled water, and then harvested at the indicated time. Total RNA was precipitated and subjected to northern blotting (upper panel). Ratios of each transcript relative to ACT1 mRNA were calculated and summarized by a graph (lower panel). (C) Examination of RP mRNA levels in amino acid-depleted media. Exponentially growing cells as used in (B) were transferred into SC—his, trp media and harvested at the indicated time. Northern blot was performed with RPS3 and ACT1 probes.

Gcn4p directly interacts with Rap1p. (A) Mapping the binding domain of Gcn4p responsible for Rap1p binding. A diagram of the Gcn4p truncation fused to GST is presented (left panel). A GST pull-down assay was performed with His₆–Rap1 and GST or GST–Gcn4p variants overexpressed in bacteria. Bead-bound GST or GST fusion proteins were detected by CBB staining and co-precipitated His₆–Rap1 was examined by western blot with an anti-Rap1p antibody (NLS, nuclear localization signal). (B) Mapping the binding domain of Rap1p responsible for Gcn4p binding. A map of the truncation mutants of His₆–Rap1p is presented in the upper panel. Total input lysate from E. coli, and bead-bound GST and GST–Gcn4 were observed by CBB staining. Input and co-purified His₆–Rap1p variants were examined by western blot with anti-His₆ antibody (Tox, toxic domain; Act, activation domain; Sil, silencing domain). (C) In vivo association of exogenously expressed Gcn4–myc₇ and Rap1p–FLAG. Gcn4p–myc₇ and Rap1p–FLAG were expressed in the BY4741 strain by a galactose induction method as described in the . Immunoprecipitation was performed with or without 10 μg of DNaseI and anti-FLAG antibody. The lysate and co-precipitated proteins were examined by western blotting with anti-FLAG and anti-myc antibodies. (D) Co-immunoprecipitation with endogenous Gcn4p and Rap1p. Total protein lysates were prepared from JS143-7D (GCN4) and YJK102 (GCN4-HA₃) after treatment with 3-AT (80 mM). Half of the 3-AT-treated sample was incubated with 10 units of alkaline phosphatase in order to examine the phosphorylation-dependent association. To exclude chromosomal DNA-dependent association, immunoprecipitation was performed in the presence of EtBr (50 μg/ml). Immobilized and co-precipitated proteins were detected by western blot with anti-HA and anti-Rap1p antibodies, respectively.

Gcn4p is recruited to RP promoters. (A) Promoter occupancy of RP genes by Gcn4p. After 3-AT (80 mM) treatment, the cultivated cells (YJK102) were harvested and immunoprecipitation was performed with an anti-HA antibody. Subsequently, co-immunoprecipitated chromatin DNA with Gcn4–HA₃ was amplified by semi-qPCR using primers specific for each promoter region. Ratios of IP relative to input signal were evaluated. Average results from more than three independent experiments are summarized in a graph. ChIP was performed three times using independent colonies (^**P<0.005; ^*P<0.05). (B) Mapping the RP promoter region responsible for Gcn4p recruitment. ChIP was performed as in (A). The co-purified chromosomal DNA was subjected to semi-qPCR. The number of PCR products refer to the following sequences: (I) −1576 to −1277 bp; (II) −365 to −2 bp; (III) +426 to +723 bp from translation start site (+1) of RPS3.

Gcn4p inhibits the transcription of RP genes through direct interaction with Rap1p. (A) Binding affinity of wild-type Gcn4p and the S242L mutant to Rap1. GST, GST–Gcn4_WT and GST–Gcn4_S242L induced in bacteria were loaded onto GST bead followed by binding with recombinant His₆–Rap1. Purified GST proteins were examined by CBB staining and co-precipitated His₆-Rap1 was analysed by western blot with an anti-Rap1p antibody. (B) Ectopically expressed Gcn4p represses the transcription of RP genes. The BY4741Δgcn4 strain harbouring myc₇, Gcn4_WT–myc₇ and Gcn4_S242L–myc₇ were cultured in SRG media for galactose induction. The expression of Gcn4_WT–myc₇ and Gcn4_S242L–myc₇ was examined by western blotting using an anti-c-myc antibody. For the detection of RP gene transcripts, northern blot analysis was performed with specific probes as indicated. (C) Gcn4_WT–myc₇, Gcn4_S242L–myc₇ and myc₇ were overexpressed using a galactose-inducible promoter as described in (B). Subsequent ChIP analysis was performed with a c-myc antibody followed by semi-qPCR with a specific probe against each promoter region as indicated. The experiments were performed in triplicate with independent colonies (^**P<0.005; ^*P<0.05). (D) Three exponentially growing strains harbouring different C-terminal tri-HA-tagged Gcn4p variants (YJK102, WT; YJK125, S242L; YJK126, GΔC) were treated with 3-AT (80 mM) for 2 h. Protein lysates from these cells were subjected to Co-IP with anti-HA antibody. Proteins in input lysates and Co-IP fractions were detected with specific antibodies as indicated. (E) The same strains used in (D) and the GCN4 deletion strain (YJK101, Δ) were cultivated with or without 3-AT (80 mM). After 1 h, cells were harvested and total RNA was precipitated. The RNA samples were subjected to northern blotting with specific probes for RPS3, RPS11B, RPL19B, TRP3 and ACT1. (F) The three strains containing Gcn4p variants were treated with 3-AT (80 mM) for 1 h. Lysates were extracted and subjected to ChIP analysis with an HA antibody. Co-immunoprecipitated chromatin DNA was examined by semi-qPCR with primers for the promoter region of the indicated genes or for the coding sequence of POL1.

Gcn4p influences recruitment of Esa1p to RP promoters. (A) Expression level of factors involved in RP transcription. The GCN4 wild-type and deletion strain containing Ifh1–HA₃ (YJK115 and YJK116), Fhl1–HA₃ (YJK113 and YJK114), Sfp1–HA₃ (YJK119 and YJK120), Esa1–HA₃ (YJK105 and YJK106), TAP–Rap1 (YJK109 and YJK110) and Gcn4–HA₃ (YJK102) were treated with 3-AT (80 mM) and harvested at the indicated times (details of strains are shown in ). Whole-cell lysates from these strains were subjected to western blotting with specific antibodies against HA, TAP and Pgk1p. (B–F) Promoter occupancies of RP genes by factors involved in RP transcription. The same strains expressing the proteins as in (A) were cultivated with or without 3-AT (80 mM). After a 1 h incubation, the cells were harvested for preparation of input lysates. ChIP assay was performed with anti-HA or anti-TAP antibodies, and amount of co-immunoprecipitated chromatin DNA was investigated by semi-qPCR with primers against the promoter regions of RPS3, RPS11B, RPL19B, and against the ORF region of POL1. JS143-7D and YJK101 strains were used as a negative control for the IP experiment. Analysis was performed in triplicate and averages of IP/input signal are summarized in a graph (^**P<0.005; ^*P<0.05).

Gcn4p inhibits Rap1p–Esa1p binding. (A) Esa1p binds directly to Rap1p in vitro. GST pull-down analysis using recombinant His₆–Rap1, and GST or GST–Esa1 proteins was performed. Proteins input or immunoprecipitated were investigated by western blot or CBB staining. (B) Whole-cell lysates of the BY4741 strain containing indicated combinations of ectopically expressed HA–Esa1 and/or Rap1–FLAG were subjected to co-immunoprecipitation with anti-FLAG or anti-HA antibodies, respectively. Input lysates and precipitated immuno-complexes were examined by western blotting with antibodies against the corresponding epitope tags. (C–E) Association of Esa1p with Rap1p was decreased by Gcn4p under amino-acid starvation. (C) BY4741 cells containing Rap1–FLAG, HA–Esa1 and myc₇ or Gcn4-myc₇ were harvested after galactose induction. FLAG–Rap1 was immobilized with an anti-FLAG antibody and protein-A agarose, followed by western blotting with antibodies against FLAG, HA and c-myc to detect proteins in input lysates and precipitated fractions. (D) The BY4741 (GCN4) and BY4741Δgcn4 (Δgcn4) strains harbouring the indicated combinations of HA–Esa1 and Rap1–FLAG were cultivated in SRG media for galactose induction, and treated with 10 μg/ml SM or drug vehicle alone. Cells were harvested at the indicated times. Associations of proteins were analysed by co-immunoprecipitation with HA antibody, and input lysates and co-immunoprecipitated proteins were detected with anti-FLAG, anti-HA, anti-Gcn4p and anti-Pgk1p antibodies. (E) GCN4 wild-type and deletion strains harbouring N-terminal TAP-tagged Esa1 (YJK127 and YJK128) were cultivated and treated with 3-AT (80 mM) or drug vehicle alone. After 2 h of incubation, cells were harvested and protein lysates were subjected to pull-down analysis with IgG sepharose beads. Proteins in input lysates and precipitated fractions were examined by western blot with antibodies against Rap1p, TAP and Pgk1p. JS143-7D and YJK101 strains were used as the negative control. (F, G) Competition between Esa1p and Gcn4p for Rap1p binding. (F) A GST pull-down analysis was performed with lysates from cells overexpressed His₆–Rap1, GST or GST–Esa1 with increasing amounts of Gcn4–His₆. GST or GST–Esa1 loaded onto beads was examined by CBB staining and the level of co-purified His₆–Rap1 was detected by western blotting with an anti-His₆ antibody. (G) An in vitro binding competition assay was performed as in (A) with some modifications. After the second binding reaction of GST pull-down analysis with GST–Esa1 and His₆–Rap1 (500 μg, 5.36 nmol), the beads were washed with 1 ml PBS and an increasing amount of purified Gcn4–His₆ (100 μg, 3.11 nmol; 300 μg, 9.34 nmol; 500 μg, 15.56 nmol) or BSA (1 mg, 15.06 nmol) was added into the binding mixture. Following a 1 h incubation at RT, the eluted fraction was separated by centrifugation. His₆–Rap1 in elution and bead-bound fractions was examined by western blot with an anti-His₆ antibody.

Dual roles of Gcn4p in response to amino-acid starvation. (A) Histone H4 acetylation on RP promoter regions under amino-acid starvation. JS143-7D (GCN4) and YJK101 (Δgcn4) were grown to an early log phase, treated with 3-AT (80 mM) and harvested after 1 h. ChIP was then performed with antibodies specific for acetyl-histone H4 (AcH4) or rabbit pre-immune serum (IgG). The semi-qPCR analysis was performed to examine co-immunoprecipitated chromatin DNA with primers specific for the promoter region of RPS3, RPS11B and RPL19B, or for the coding sequence of POL1 CDS. Experiments were performed in triplicate with independent colonies (^**P<0.005; ^*P<0.05). (B) Schematic diagram for the roles of Gcn4p under amino-acid starvation.