MATERIALS AND METHODS Expression, purification and crystallization of human Rpb4/Rpb7 Co-expression in Escherichia coli of the human Rpb4/Rpb7 5 complex was achieved by using a bicistronic construct based upon pGEX-2TK (Amersham Biosciences) in which the Rpb4 subunit was fused to a glutathione-S-transferase tag that was cleavable by thrombin, whereas the Rpb7 subunit was expressed as an untagged protein (18). E.coli BL21(DE3) 10 cells were transformed under the control of ampicillin and, after induction at OD600 between 0.6 and 0.9, cell cultures were grown at 28 C overnight. The cells were harvested by centrifugation and resuspended in 20 mM Tris–HCl, pH 7.9, 300 mM potassium acetate, 7 mM magnesium acetate, 1 mM 15 EDTA, 10 mM DTT, 10% glycerol (buffer P300), supplemen- ted with lysozyme at 1 mg/ml, Benzonase (0.1U/ml) and 1 mM 4-(2-aminoethyl)benzenesulphonyl ﬂuoride. Cell lysis was performed by sonication and the resulting lysate clariﬁed by centrifugation and ﬁltration. All the chromatographic steps 20 were performed at 4 C. The crude extract was bound in batch to glutathione sepharose beads (Amersham Biosciences) and the beads were extensively washed with buffer P300. The protein was recovered from the beads by cleavage with throm- bin overnight at 4 C. In preparation for anion exchange chro- 25 matography, the supernatant was dialyzed against buffer A [20 mM Tris–HCl, pH 8.0, 1 mM EDTA, 10% glycerol and 10 mM b-mercaptoethanol] containing 100 mM NaCl. The dialyzed protein solution was loaded onto a Resource Q col- umn (Amersham Biosciences) using an A¨ KTA FPLC appar- 30 atus (Amersham Biosciences) and eluted with a linear gradient from 100 mM to 1 M NaCl in buffer A. After concentration, the sample was applied to a Superdex-200 10/30 gel ﬁltration column (Amersham Biosciences) equilibrated with 20 mM MES, pH 6.5 and 100 mM NaCl. Pooled peak fractions 35 were ﬂash frozen in liquid nitrogen and stored at 80 C until further use. For crystallization the protein was concentrated to 15 mg/ml in 20 mM PIPES, pH 6.8, 100 mM NaCl and 15% glycerol. Crystals were grown at 18 C by vapour diffusion 40 in hanging drops, against a well solution containing 100 mM PIPES pH 7.4, 25% polyethylene glycol (MW 4000) and 15% glycerol. Most of the crystals grew as clusters of thin plates, but occasionally a few small chunky crystals could be obtained. 45 Structure determination and refinement The crystals were ﬂash-frozen directly from the drop and X-ray data collected at 100 K using a MAR image plate detector mounted on a Rigaku RU-H3R rotating anode X-ray generator equipped with OSMIC focusing mirrors 50 (CuKa radiation; l ¼ 1.54 s). The diffraction data were pro- cessed with MOSFLM and programs of the CCP4 suite (19). The crystals are in space group P1 with four heterodimers in the asymmetric unit (Table 1). A search model for molecular replacement was constructed from the yeast Rpb4/Rpb7 com- 55 plex (29% sequence identity for Rpb4, 37% sequence identity for Rpb7) (16). The positions of the four copies of the hetero- dimer in the asymmetric unit were determined with the pro- gram PHASER (20), and alternating cycles of reﬁnement with Nucleic Acids Research, 2005, Vol. 33, No. 19 6437 Table 1. Data collection and refinement statistics Space group Cell parameters (A˚ ) Resolution (A˚ ) Rmerge (%) Multiplicity Completeness (%) I/s Rwork (%) Rfree (%) R.m.s.d. bonds (A˚ ) R.m.s.d. angles (degrees) Number of protein atoms Number of water molecules P1 a ¼ 45.69, b ¼ 83.19, c ¼ 99.31, a ¼ 85.53 , b ¼ 81.80 , g ¼ 81.77 43.3–2.7 (2.85–2.7) 6.9 (39.4) 3.4 (3.4) 95.3 (93.8) 14.5 (3.4) 23.1 26.6 0.008 1.4 9236 54 Highest resolution shell values shown in parentheses. CNS (21) and model building with O (22) resulted in the ﬁnal model. The four copies of the Rpb7 subunit make different 60 crystal contacts and adopt two slightly different conforma- tions. The largest differences are located in the A1–K2 and the A3–A4 loops (Figures 1 and 2) with residues 60–63 dif- fering in position by >5 s. Tight restraints between pairs of heterodimers were applied during the reﬁnement procedure. 65 The reﬁned model has 98.9% of the residues in allowed or additionally allowed regions of the Ramachandran plot and no residues in disallowed regions. Residue 172 of Rpb7 and resi- dues 1–13 and 142 of Rpb4 have no electron density and are presumed disordered. Crystallographic statistics are summar- 70 ized in Table 1. The coordinates have been deposited in the Protein Data Bank (entry 2C35). Expression and purification of the wild-type and mutant protein for RNA binding The wild-type human Rpb4/Rpb7 complex was expressed and 75 puriﬁed as described above. For expression of the Methano- caldococcus jannaschii heterodimer, subunits E and F were subcloned generating a bicistronic expression construct in pET21a(+) (Novagen) and co-expressed as untagged proteins in BL21(DE3) cells by inducing exponentially growing cells 80 with 0.5 mM Isopropyl-b-D-thiogalactopyranoside for 3–5 h at 37 C. Cells were lysed in P300 as described above and the lysate held at 75 C for 40 min. The thermally denatured E.coli proteins were removed by centrifugation and the supernatant incubated with DEAE-sepharose in order to remove nucleic 85 acids. The resulting lysate preparation was loaded onto a HiLoad Superdex-75 26/60 gel ﬁltration column (Amersham Biosciences) and equilibrated with 20 mM Tris–HCl, pH 8.0 and 100 mM NaCl. Pooled peak fractions were concentrated to between 10 and 15 mg/ml, ﬂash frozen in liquid nitrogen 90 and stored at 80 C. Site-directed mutagenesis was performed using the QuikChange (Stratagene) mutagenesis kit according to the manufacturer’s instructions. After conﬁrmation of each muta- tion by sequencing, the M.jannaschii and human complexes 95 were expressed in E.coli as described above and puriﬁed fol- lowing essentially the same strategy as used for the corres- ponding wild-type proteins, with the exception that the anion exchange chromatography step was omitted in the puriﬁcation of the human Rpb4/7 mutants. 100