Entrevistas

4.3.1 Expression and purification of the MED7/MED10/MED21/MED30 middle module complex

Plasmids containing MED7/MED10/MED21 and MED31 were cotransformed into BL21 DE3 RIL and expressed as described in 4.1.5. For purification of expressed pro- teins cells were thawed at 30◦ C and lysed by sonication with a Branson sonifier 250

and a flat 1

2” working tip for 15 min 20 % duty time and 40 % output. The lysate was

then centrifuged for 30 min at 4◦ C and 16000 rpm in a SS34 rotor. The cleared lysate

was loaded onto a preequilibrated 1 ml HisTrap HP (Amersham) column washed with 20 mM of lysis buffer and eluted stepwise with lysis buffer containing 20, 40, 70 and 200 mM imidazole. Protein elution was monitored by absorption at 280 nm and Brad- ford reagent (Biorad). Eluted fractions were analyzed by 17 % SDS-PAGE. The main fractions of the 70 mM imidazole elution were diluted with buffer A and loaded on a MonoQ 10/100 GL (Amersham) anion exchange chromatography column and eluted with a 20 column volume linear gradient of buffer A containing 50 mM to 1 M NaCl. The main fractions were analyzed by 17 % SDS-PAGE, pooled and concentrated in Amicon Ultra centrifugal devices with 10 KDa molecular weight cut off. The con- centrated samples were loaded onto a Superose 6 10/300 GL (Amersham) gelfiltration column equilibrated with Buffer B. Peak fractions were analyzed by 17 % SDS-PAGE, concentrated to 4.5 mg/ml and used for crystallization setups.

4.3.2 Reconstitution of a MED4/MED7/MED9/MED10/MED21/MED30 middle module complex

The 40 mM His Trap elution of MED7/MED10/MED21/MED30 was mixed in a 1 to 1.5 ratio with ammonium sulfate purified MED4/MED9 (4.2.3) and the complex was assembled for 1 h at 20◦ C. After 1h the conductivity was adjusted to less than 100

mS/cm3

by the addition of buffer A and the sample was loaded on a MonoQ 10/100 GL (Amersham). Elution was as for MED7/MED10/MED21/MED30. Pure fractions were concentrated in Amicon Ultra centrifugal devices (10 KDa molecular weight cut off) and applied to a Superose 6 10/300 GL (Amersham) gelfiltration column preequi- librated with buffer B. Peak fractions were analyzed by SDS-PAGE, concentrated to a final concentration of 5 mg/ml and used for Crystallization setups as well as for Pol II middle module complex assembly.

4.3.3 Assembly of a Pol II/middle module complex

For reconstitution of a middle module Pol II complex 10 subunit Pol II, Rpb4/Rpb7 and the purified middle module complex are assembled. Pol II and Rpb4/Rpb7 were obtained from Stefan Benkert. Buffers of 10 subunit Pol II and middle module were exchanged to Pol II buffer using Amicon Ultrafree-MC centrifugal devices with 100 and 10 KDa cut off, respectively. Rpb4/Rpb7 were obtained frozen in Pol II buffer and thawed on ice. 10 subunit Pol II, Rpb4/Rpb7, and middle module were combined and incubated on a rotating wheel at 20◦ C for 1 h prior to gelfiltration on Superose

6 10/300 GL (Amersham). 5 fold excess of Rpb4/Rpb7 and 3 fold Excess of middle module were used the total volume was adjusted to 200µl. Peak fractions were ana-

lyzed by 17 % SDS-PAGE, fractions containing the complex were concentrated to 200

µl and reloaded onto a Superose 6 gelfiltration column. The peak fractions were again

buffer description

lysis buffer 150 mM NaCl, 20 mM Tris pH 8.5 (4◦ C), 10 mM _β-

mercaptoethanol, 1 mM PMSF, 0 to 200 mM imidazole MonoQ buffer 50 mM to 1 M NaCl, 20 mM Tris pH 8.5 (4◦ C), 10 mM

β-mercaptoethanol

ammonium sulfate at room temperature saturated solution of ammonium sul- fate

middle module buffer 150 mM NaCl, 20 mM Tris pH 8.5 (4◦ C), 10 mM _β-

mercaptoethanol

Pol II buffer 40 mM ammonium sulfate, 5 mM Hepes pH 7.25 10 µM

ZnCl2, 10 mM DTT

Table 13: Buffers used for the purification and reconstitution of the middle module

4.3.4 Gst-CTD pull-down

Gst-CTD pull-downs were done to test binding of the six-subunit middle module com- plex to the CTD of Pol II. Gst fusion proteins of the CTD repeats only and the CTD containing the linker domain of Rpb1 were used. Both Gst-CTD constructs as well as a Gst only control were expressed as described in 4.1.5. The cells were resuspended in 50 HGN100 buffer and lysed by sonication as described above. After two 30 min- utes centrifugation steps (SS34, 16000rpm, 4◦ C). 500_µl of prewashed Gst sepharose

(CL4B, Amersham) was added to the cleared lysate. Gst fusion protein was bound to the beads during 4 hours at 4◦ C on a rotating wheel. The slurry was decanted

into plastic columns (Qiagen) and washed with 20 ml of HEGN100 buffer. Amounts and purity of the bound protein was analyzed by Bradford and SDS-PAGE. Protein amounts were equalized by diluting with empty Gst sepharose to a final concentra- tion of 0.5µg/µl beads. As a positive control the CTD interacting domain of Pcf11

(residues 1 to 140) was expressed and purified according to (Meinhart and Cramer, 2004). Pcf11 and the reconstituted middle module was dyalized against HEGN100 buffer and incubated with 30µl Gst-CTD beads over night. The beads were washed

for four times with HEGN100, boiled in SDS sample buffer and analyzed by SDS- PAGE and western blot. The HisProbe system (pierce) was used to detect His-tagged proteins.

buffer description

HEGN100 10% glycerol, 100µM EDTA, 20 mM Hepes pH 7.6, 0.1 %

NP40, 100 mM KCl, 1mM PMSF, 5mM DTT BSA 10 mg/ml solution (Biolabs)

Table 14: Buffers used for the Gst-CTD pull-down