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. 2009 Sep 29;106(39):16734-9.
doi: 10.1073/pnas.0905103106. Epub 2009 Sep 11.

Mediator complex association with constitutively transcribed genes in yeast

Suraiya A Ansari  1 , Qiye He, Randall H Morse
Affiliations

Mediator complex association with constitutively transcribed genes in yeast

Suraiya A Ansari et al. Proc Natl Acad Sci U S A. .

Abstract

Mediator is a large, multisubunit complex that is essential for transcription of mRNA by RNA Pol II in eukaryotes and is believed to bridge transcriptional activators and the general transcription machinery. However, several recent studies suggest that the requirement for Mediator during transcriptional activation is not universal, but rather activator dependent, and may be indirect for some genes. Here we have investigated Mediator association with several constitutively transcribed genes in yeast by comparing a yeast strain that harbors a temperature-sensitive mutation in an essential Mediator subunit, Srb4, with its wild-type (WT) counterpart. We find modest association of Mediator with constitutively active genes and show that this association is strongly decreased in srb4 ts yeast, whereas association with a nontranscribed region or repressed gene promoters is lower and unaffected in the mutant yeast. The tail module of Mediator remains associated with ribosomal protein (RP) gene promoters in srb4 ts yeast, while subunits from the head and middle modules are lost. Tail module association at Rap1-dependent gene promoters is lost in rap1 ts yeast, indicating that Rap1 is required for Mediator recruitment at these gene promoters and that its recruitment occurs via the tail module. Pol II association is also rapidly and severely affected in srb4 ts yeast, indicating that Mediator is directly required for pol II association at constitutively transcribed genes. Our results are consistent with Mediator functioning as a general transcription factor in yeast.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Mediator associates specifically with constitutively transcribed genes. (A and B) Association of Srb5 from the head module of Mediator with the core promoter and UAS of the indicated genes was analyzed by ChIP. WT and srb4 ts mutant strains expressing Myc13-tagged Srb5 were grown at 25 °C and shifted to 37 °C for 45 min before carrying out ChIP. ChIPed DNA was analyzed by real time PCR. The bars represent log2 of the IP/input ratios for the indicated genes, normalized to log2 of IP/input for ChrV, as described in Materials and Methods. (C and D) Association of Rgr1 from the middle module of Mediator in WT and srb4 ts yeast strains expressing TAP-tagged Rgr1 was analyzed as described for Srb5. Error bars represent standard deviation. Each experiment was performed 3–4 times.
Fig. 2.
Fig. 2.
Srb5 association is selectively affected at RP genes in rap1 ts yeast. Association of Srb5 from the head module of Mediator was analyzed in rap1 ts and WT yeast at the core promoter (A) and UAS regions (B) of the indicated genes. WT and rap1 ts mutant yeast strains expressing Myc13-tagged Srb5 were grown at 25 °C and shifted to 37 °C for 1 h before carrying out ChIP. ChIPed DNA was analyzed by real time PCR. The bars represent log2 of IP/input ratios for the indicated genes, normalized to log2 of IP/input ratios for ChrV. Error bars represent standard deviation. Each experiment was performed 3–4 times.
Fig. 3.
Fig. 3.
The association of Gal11 remains unaffected in srb4 ts mutant strain at nonpermissive temperature but is selectively affected at RP gene promoters in rap1 ts mutant yeast. (A–D) Association of Gal11 from the tail module of Mediator with the core promoter and UAS of the indicated genes was analyzed by ChIP. WT, srb4 ts, and rap1 ts mutant strains expressing Myc13-tagged Gal11 were grown at 25 °C and shifted to 37 °C for 45 min (srb4 ts and WT) or 1 h (rap1 ts and WT) before carrying out ChIP. ChIPed DNA was analyzed by real time PCR. The bars represent log2 of IP/input ratios normalized to log2 of IP/input ratios for ChrV. Error bars indicate standard deviation. Each experiment was performed 3–4 times.
Fig. 4.
Fig. 4.
Rapid loss of Mediator and pol II association at constitutively transcribed genes in srb4 ts mutant yeast at nonpermissive temperature. Association of Srb5 from the head module of Mediator (A and B) and of the Rpb3 subunit of pol II (C and D) with the core promoters of the indicated genes was determined by ChIP in WT and srb4 ts yeast at 25 °C or after 2 min at 34 °C, as indicated. ChIPed DNA was analyzed by real time PCR. The bars represent log2 of IP/input ratios for the indicated genes normalized to log2 of IP/input ratios for the tRNA gene promoter, tQ(UUG)C. Error bars indicate standard deviation. Each experiment was performed 3–4 times.
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