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. 1997 Jun 10;94(12):6075-8.
doi: 10.1073/pnas.94.12.6075.

Evidence for a mediator cycle at the initiation of transcription

J Q Svejstrup  1 , Y Li, J Fellows, A Gnatt, S Bjorklund, R D Kornberg
Affiliations

Evidence for a mediator cycle at the initiation of transcription

J Q Svejstrup et al. Proc Natl Acad Sci U S A. .

Abstract

Free and elongating (DNA-bound) forms of RNA polymerase II were separated from yeast. Most cellular polymerase II was found in the elongating fraction, which contained all enzyme phosphorylated on the C-terminal domain and none of the 15-subunit mediator of transcriptional regulation. These and other findings suggest that mediator enters and leaves initiation complexes during every round of transcription, in a process that may be coupled to C-terminal domain phosphorylation.

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Figures

Figure 1
Figure 1
Mediator exchange between RNA polymerases. Exchange was for 10 min (A) or 20 min (B, separate experiment from A) by slow rotation at room temperature between free RNA polymerase II holoenzyme (0.1–0.5 μg) and hexahistidine-tagged core RNA polymerase II bound to Ni2+-agarose beads (+) or beads with no bound polymerase (−). Following sedimentation in a microcentrifuge for 2 min at 5,000 rpm, supernatant (Sup) and beads (Pel) were analyzed by SDS/PAGE (10% gel) and immunoblotting with antibodies against the proteins indicated. The Rpb1 subunit of the polymerase bound to beads was partly N-terminally degraded, resulting in a double band, which served to identify the core polymerase. Binding of core polymerase (25–50 μg in 40–150 μl) to beads (Qiagen) was in 40 mM Hepes, pH 7.6/20% glycerol/5 mM 2-mercaptoethanol/0.25 mg/ml bovine serum albumin/protease inhibitors (12) by slow rotation for 45 min at 4°C. Following several washes by centrifugation and resuspension in 200 mM Hepes, pH 7.6/20% glycerol/5 mM 2-mercaptoethanol/0.25 mg/ml bovine serum albumin/0.2% Tween-20/0.01% Nonidet P-40/10 mM imidazole/protease inhibitors, beads were resuspended in 100 μl of the same buffer. Exchange was performed with 10 μl of beads (2–5 μg of bound polymerase) in 40 mM Hepes, pH 7.6/150 mM potassium acetate/5 mM magnesium acetate/10% glycerol/5 mM 2-mercaptoethanol/0.1 mg/ml bovine serum albumin/0.06% Tween-20/0.003% Nonidet P-40/3 mM imidazole/protease inhibitors.
Figure 2
Figure 2
Displacement of mediator but not TFIIF from RNA polymerase II holoenzyme by anti-CTD antibodies. Holoenzyme (100 μg) was incubated with 8WG16 monoclonal antibodies immobilized on protein A–Sepharose beads in 20 mM Tris⋅acetate, pH 7.8/20% glycerol/200 mM potassium acetate/0.01% Nonidet P-40/0.1 mM EDTA, as described (13) for 4 h at 4°C. The beads were removed by sedimentation in a microcentrifuge for 5 min at 5,000 rpm, and starting material (holopol II) and supernatant proteins (mediator) were compared by SDS/PAGE and silver staining. The top portion of a 10% gel is shown. Protein components of the holoenzyme preparation are indicated on the left. In addition to the Tfg1 subunit of TFIIF, the two smaller Tfg2 and Tfg3 subunits failed to appear in the supernatant (mediator) fraction, as did the subunits of RNA polymerase II.
Figure 3
Figure 3
Components of free and DNA-bound RNA polymerase II complexes. Following separation as described, free (Sup) and DNA-bound (Pel) fractions were analyzed by SDS/PAGE (6% gel) and immunoblotting to reveal the largest polymerase subunit Rpb1 (12CA5 antibody) and the mediator component Srb4. Relative amounts of phosphorylated Rpb1 (Pol II0) and Srb4 in the free and DNA-bound fractions are indicated above the bars in the graph at the bottom. The relative amounts of phosphorylated (II0) and unphosphorylated (IIA) Rpb1 varied from one preparation to another, possibly due to phosphatase action, but the phosphorylated form was always found exclusively in the DNA-bound fraction.
Figure 4
Figure 4
Effect of TFIIS on recovery of RNA polymerase II in DNA-bound fraction. Free and DNA-bound RNA polymerase II complexes were separated as described, except that the whole-cell extract (50 μl, 20–30 mg protein/ml) was treated with TFIIS (gift from A. M. Edwards, McMaster University, Hamilton, Ontario, Canada) in the amounts indicated in a volume of 100 μl containing 50 mM Tris⋅Cl, pH 7.5/60 mM ammonium sulfate/10 mM magnesium chloride/10% glycerol/10 μM zinc sulfate for 30 min at 30°C, followed by chilling in ice, adjustment of potassium acetate concentration, and PEI precipitation. The amount of Rpb1 in the PEI pellet was determined by SDS/PAGE (10% gel) and immunoblotting. Phosphorylated and unphosphorylated forms of Rpb1 were not resolved in this percentage gel.
Figure 5
Figure 5
Proposed mediator cycle. RNA polymerase II (Pol II), associated with mediator and general initiation factors (TFII’s), interacts with DNA (thick horizontal line) at the start site of transcription (arrow above line). TFIIH phosphorylates the CTD (wavy line), concomitant with mediator release and the initiation of transcription. Dephosphorylation of the CTD and rebinding of mediator complete the cycle. NTP’s, nucleoside triphosphates.
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