Sw60 swinging bucket rotor

Pol II was phosphorylated with DYRK1A kinase as described above. A nucleic acid scaffold for transcribing Pol II was assembled by mixing equimolar amounts of DNA (EC-phf3-template) and RNA (RNA50) in a BioRad T100 Thermal Cycler heated to 95 °C and cooled in 0.1 °C/s increments until 4 °C was reached. For sucrose gradient ultracentrifugation, the Pol II-EC was assembled by incubating 60 pmol Pol II with a 2-fold molar excess of DNA/RNA for 10 min on ice, followed by 10 min at 30 °C, and another 10 min at 30 °C after adding a 4-fold molar excess of non-template DNA (EC-phf3-nontemplate) to generate a transcription bubble. A 4-fold molar excess of PHF3 or TFIIS^M/TFIIS^M+TFIIF was incubated with Pol II-EC for 20 min at 25 °C, followed by addition of the 4-fold molar excess of the competitor (TFIIS^M/TFIIS^M+TFIIF or PHF3 respectively) for 20 min at 25 °C. 10–30% sucrose gradients were prepared using a gradient mixer (Gradient Master 108; BioComp Instruments). Pol II complexes were applied on top of the gradient followed by ultracentrifugation 105169 g in a SW60 swinging bucket rotor (Beckman Coulter) for 16 h at 4 °C. 80 µl fractions were collected carefully from top to the bottom of the tube and analyzed by Western blotting.

To investigate the binding of TFIIF and Paf1C to Pol II, analytical sucrose gradient ultracentrifugation was carried out. A sucrose gradient was generated by mixing equal volumes of light solution (10 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM DTT, 2 mM MgCl₂, 10 μM ZnCl₂, 10% (w/v) sucrose) and heavy solution 2 (10 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM DTT, 2 mM MgCl₂, 10 μM ZnCl₂, 30% (w/v) sucrose) using a gradient mixer (Gradient Master 108; BioComp Instruments).
Reconstituted Pol II-Paf1C-TFIIS EC was divided into portions. The first portion was incubated with a 1.8-fold molar excess of TFIIF54 (link), while the other portion was incubated with buffer D (10 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM MgCl₂, 2 mM DTT, 10 μM ZnCl₂) as a control. Next, the reconstituted complexes were applied on top of the gradient. After ultracentrifugation at 32,000 r.p.m. in a SW60 swinging bucket rotor (Beckman Coulter) for 16 h at 4 °C, 200 μl fractions were collected by pipetting carefully from top to bottom of the tube before analysing them by SDS–PAGE and native PAGE. The assay was repeated but starting from a Pol II-TFIIF complex and then incubating with excess Paf1C-TFIIS.