Activator-independent functions of the yeast mediator sin4 complex in preinitiation complex formation and transcription reinitiation

Abstract

RNA polymerase II (Pol II) Mediator plays an essential role in both basal and activated transcription. Previously, subunits of the Sin4 Mediator complex (Sin4, Pgd1, Gal11, and Med2) have been implicated in both positive and negative transcriptional regulation. Furthermore, it was proposed that this subcomplex constitutes an activator-binding domain. A yeast nuclear-extract system was used to investigate the biochemical role of the Sin4 complex. In contrast to previous findings, we found at least two general activator-independent roles for the Sin4 complex. First, mutations in sin4 and pgd1 destabilized the Pol II-Med complex, leading to a reduced rate and extent of preinitiation complex (PIC) formation both in the presence and absence of activators. Although reduced in amount compared with the wild type, PICs that are formed lacking the Sin4 complex are stable and can initiate transcription normally. Second, mutation of pgd1 causes partial disruption of the Sin4 complex and leads to a defect in transcription reinitiation. This defect is caused by dissociation of mutant Mediator from promoters after initiation, leading to nonfunctional Scaffold complexes. These results show that function of the Sin4 complex is not essential for transcription activation in a crude in vitro system but that it plays key roles in the general transcription mechanism.

FIG. 1.

Yeast Mediator. Proposed interactions within the Sin4 complex are shown (8, 16, 29).

FIG. 2.

Δpgd1 and Δsin4 mutants have a general defect in plasmid multiround transcription. PICs were formed on plasmid template for 40 min, NTPs were added to initiate transcription, and the reactions were stopped after 2.5 (A) or 40 (B) min. Transcripts were assayed by primer extension (gels). Results of typical transcription reactions with wild-type, Δpgd1, and Δsin4 extracts and with no activator (−), Gal4-AH (A), Gal4-VP16 (V), or Gal4-Gcn4 (G) are shown. Levels of activation (n-fold) are indicated below the gels. Data in the graphs are averages from at least four experiments, with the level of mutant transcription calculated as a percentage of wild-type transcription. (C) Rounds of transcription were calculated by dividing multiround transcription by single-round transcription. The results shown are an average of three experiments. (D) VP16-activated multiround transcription was assayed as in panel B with 0, 1, or 2 μl of purified Pol II-Med complex (∼0.04 pmol/μl) added to the mutant nuclear extracts (NEs).

FIG. 3.

Sin4 and Pgd1 promote efficient PIC formation. Wild-type extract and Δpgd1 (A) or Δsin4 (B) extract were incubated with Gal4-VP16 activator and plasmid template in transcription mix for the indicated times. PIC formation was assayed by adding NTPs for a single 2.5-min round of transcription.

FIG. 4.

Sin4 complex stabilizes Pol II-Med. Nuclear extracts (NEs) were incubated with protein A Dynabeads either with (+) or without (−) bound anti-Srb4 polyclonal antibody. Immunoprecipitated proteins were analyzed by SDS-PAGE and Western blotting. After subtracting nonspecific binding (−), the band intensities were normalized to that of the wild type. The ratio of Pol II-Mediator components to Srb4 is shown.

FIG. 5.

Mutants have a general defect in single-round and multiround transcription on an immobilized template. PICs were formed on an immobilized template for 40 min. As described for Fig. 2, NTPs were added to initiate transcription and the reactions were stopped after 2.5 (A) or 40 (B) min. Transcripts were assayed by primer extension (gels). Results of typical transcription reactions with wild-type, Δpgd1, and Δsin4 extracts with no activator (−), Gal4-AH (A), Gal4-VP16 (V), or Gal4-Gcn4 (G) are shown. Levels of activation (n-fold) are indicated below the gels. Data shown in the graphs are the averages from at least two experiments, with the level of mutant transcription calculated as a percentage of wild-type transcription. (C) Rounds of transcription were calculated by dividing multiround transcription by single-round transcription. The results shown are an average of two experiments.

FIG. 6.

Pgd1 and Sin4 are required for optimal PIC formation. (A) PICs were formed in the presence of Gal4-VP16 for 40 min. Promoter-bound proteins were assayed by SDS-PAGE and Western blotting. (B) The experiment described in panel A was repeated with no activator, Gal4-AH, Gal4-VP16, and Gal4-Gcn4. Mutant PIC formation is shown as a percentage of wild-type PIC formation, based on the presence of a complete PIC (including Pol II, TFIIB, TFIIH, and Mediator). The results are an average of at least two experiments. (C) Identical to panel A but showing Sin4 complex subunits. (D) Washed PICs were formed as described for panel A, and PICs were then resuspended in transcription mix plus competitor DNA. After 0 or 40 min, PICs were washed once and bound proteins were visualized by SDS-PAGE and Western blotting.

FIG. 7.

Δsin4, but not Δpgd1, nuclear extracts (NEs) can form stable Scaffold complexes. PICs (without NTPs) were formed for 40 min in the presence of the activator Gal4-VP16 and washed. To form Scaffold complexes, washed PICs were resuspended in transcription mix and initiated with NTPs (+) for 3.0 min. Washed promoter-bound Scaffold complexes were either immediately digested from the beads (0 min) or resuspended in transcription mix plus competitor DNA for 40 min before being washed and digested (40 min). For more accurate quantitation, 2× Δpgd1 and 4× Δsin4 reactions were performed. The graph compares the retention of Scaffold components in mutants to their retention in wild-type extracts at time 0. The calculations are based on at least five experiments.

FIG. 8.

Mediator is required for Scaffold function. PICs and Scaffold were formed as described in the legend to Fig. 5, except that ATP was used instead of NTPs. In lanes 1 to 3, washed-PIC transcription (trxn) was assayed. To assay Scaffold transcription in lanes 5, 7, and 9, Scaffold complexes were resuspended in transcription mix plus competitor DNA, NTPs, and Δsrb2 nuclear extract (NE) for 2.5 min. As a control, background transcription was determined in lanes 4, 6, and 8 by resuspending Scaffold complexes in transcription mix plus competitor DNA without a second nuclear extract. Scaffold-independent transcription by Δsrb2 nuclear extract was assayed in lane 10 on a template that had previously been incubated only with Gal4-VP16. For the calculations presented in the figure and text, Scaffold-specific transcription was determined by subtracting these two controls from lanes 5, 7, and 9.