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. 2000 Oct;20(20):7427-37.
doi: 10.1128/MCB.20.20.7427-7437.2000.

Saccharomyces cerevisiae transcription elongation mutants are defective in PUR5 induction in response to nucleotide depletion

R J Shaw  1 , D Reines
Affiliations

Saccharomyces cerevisiae transcription elongation mutants are defective in PUR5 induction in response to nucleotide depletion

R J Shaw et al. Mol Cell Biol. 2000 Oct.

Abstract

IMP dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo synthesis of guanine nucleotides. It is a target of therapeutically useful drugs and is implicated in the regulation of cell growth rate. In the yeast Saccharomyces cerevisiae, mutations in components of the RNA polymerase II (Pol II) transcription elongation machinery confer increased sensitivity to a drug that inhibits IMPDH, 6-azauracil (6AU), by a mechanism that is poorly understood. This phenotype is thought to reflect the need for an optimally functioning transcription machinery under conditions of lowered intracellular GTP levels. Here we show that in response to the application of IMPDH inhibitors such as 6AU, wild-type yeast strains induce transcription of PUR5, one of four genes encoding IMPDH-related enzymes. Yeast elongation mutants sensitive to 6AU, such as those with a disrupted gene encoding elongation factor SII or those containing amino acid substitutions in Pol II subunits, are defective in PUR5 induction. The inability to fully induce PUR5 correlates with mutations that effect transcription elongation since 6AU-sensitive strains deleted for genes not related to transcription elongation are competent to induce PUR5. DNA encompassing the PUR5 promoter and 5' untranslated region supports 6AU induction of a luciferase reporter gene in wild-type cells. Thus, yeast sense and respond to nucleotide depletion via a mechanism of transcriptional induction that restores nucleotides to levels required for normal growth. An optimally functioning elongation machinery is critical for this response.

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Figures

FIG. 1
FIG. 1
(A) Time course of IMPDH mRNA induction by 6AU. Yeast strain DY103 was diluted to an OD600 of 0.1 in SC-Ura and grown to an OD600 of ≈0.5 at 30°C. The culture was split, and 6AU (75 μg/ml) was added to half; at the indicated times, RNA was harvested and subjected to Northern blotting. (B) Quantitation of Northern blot in panel A. (C) Underivatized uracil does not induces PUR5 mRNA synthesis. Cells were treated with 6AU or uracil (each at 75 μg/ml); RNA was harvested and subjected to Northern blotting.
FIG. 2
FIG. 2
IMPDH mRNA induction is derived largely from the PUR5 gene. Strains lacking YAR073W and PUR5 (ABGG10; A, lanes 1 to 6), PUR5 (DY731; A, lanes 7 to 12), YAR073W (DY732; A, lanes 13 to 18), or YML056C (DY743; B, lanes 7 to 12) were treated with 6AU (75 μg/ml) for the indicated times; RNA was isolated and subjected to Northern blotting. A control strain (DY741; B, lanes 1 to 6) otherwise isogenic to DY743 was also analyzed. Filters were quantitated by phosphorimaging and plotted (C).
FIG. 3
FIG. 3
Induction of PUR5 transcription by mycophenolic acid. Yeast strains DY103 (wt [wild type]) and DY108 (rpb2-10, dst1) were treated with mycophenolic acid (MPA; 15 μg/ml); RNA was isolated and subjected to Northern blotting. Results were quantitated by phosphorimaging and plotted (B).
FIG. 4
FIG. 4
Guanine suppresses PUR5 induction by 6AU. Cultures of DY103 were grown for 30 min in medium containing (lanes 3, 6, 9, and 12) or lacking (lanes 1, 2, 4, 5, 7, 8, 10, and 11) 1 mM guanine; 6AU (75 μg/ml) was added; samples were withdrawn at the indicated times and analyzed by Northern blotting. A control culture lacking guanine and 6AU was also tested (lanes 1, 4, 7, and 10). Filters were phosphorimaged; the data are plotted in panel B.
FIG. 5
FIG. 5
6AUs elongation mutants fail to induce PUR5 transcription. Strains DY103 (RPB2,DST1), DY105 (rpb2-10), DY106 (dst1), and DY108 (rpb2-10, dst1) were treated with 6AU (75 μg/ml) for the indicated times. RNA was prepared, subjected to Northern blotting (A), and quantitated by phosphorimaging (B). Only the data for drug-treated cells are plotted presented in panel B.
FIG. 6
FIG. 6
PUR5 induction in 6AUs strains mutated in Pol II. Strains DY760 (RP021), DY761 (rpb1-221), DY190 (RP021), and DY2050 (rpo21-18) were grown in the presence or absence of 6AU (75 μg/ml), and RNA was prepared for Northern blotting at the indicated times. Results were quantitated by phosphorimaging and plotted.
FIG. 7
FIG. 7
6AUs mutants in PPR1 and SNZ/SNO can induce PUR5. The experiment described for Fig. 6 was repeated with strains DY746 (Δppr1), DY742 (PPR1), MW980 (Δsno1,2,3, Δsnz1,2,3), MW1072 (SNO+, SNZ+), and MW926 (Δsnz1).
FIG. 8
FIG. 8
PUR5 induction in the rpb1-1 strain. Strains Z196 (RP021) and Z460 (rpb1-1) were grown in the presence or absence of 6AU (75 μg/ml), and RNA was prepared for Northern blotting at the indicated times. Results were quantitated by phosphorimaging and plotted.
FIG. 9
FIG. 9
PUR5 sequences confer 6AU inducibility on a luciferase reporter gene. Strains containing (PUR5) or lacking (Δpur5) PUR5 and harboring a reporter plasmid containing 800 bp of DNA of PUR5 sequence upstream of the firefly luciferase coding sequence were treated with 6AU (75 μg/ml) and frozen at the indicated times. Extracts were prepared and assayed for luciferase activity in a luminometer. Data for each strain were normalized to the maximal amount of relative light units obtained over the time course. Experiments were performed in triplicate, and the means ± standard deviations (error bars) were calculated and plotted.
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