Computational protocol: Promoter Recognition by a Complex of Spx and the C-Terminal Domain of the RNA Polymerase α Subunit

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Protocol publication

[…] “Reduced” Spx, in which residue Cys10 was replaced by serine (C10S Spx), was overexpressed in E. coli and purified by Ni2+-NTA affinity column chromatography as previously described . Purified, cleaved Spx was dialyzed into 25 mM PIPES, pH 6.5, 100 mM KCl for use in subsequent crystallization trials.αCTD containing residues 245 to 314 of RpoA was cloned into the IPTG-inducible PET28a overexpression vector (Invitrogen), which contains an N-terminal histidine tag and thrombin cleavage site. The αCTD-PET28a vector was transformed into Rosetta 2 competent cells (EMD Biosciences) for overexpression. αCTD was purified by Ni-NTA affinity chromatography (Qiagen). Following purification, the histidine tag was cleaved by incubation with thrombin protease (GE Healthsciences). Purified, cleaved αCTD was dialyzed into 25 mM Pipes, pH 6.5, 100 mM KCl. The Spx-αCTD complex was formed by mixing equimolar amounts of purified C10S Spx and αCTD. The complex was concentrated to 10 mg/ml.Crystals of the reduced C10S Spx-αCTD complex were grown by the hanging-drop vapor diffusion method. Two microliters of 0.5 mM C10S Spx-αCTD was mixed with 2 µL of reservoir solution containing 25%–30% polyethylene glycol 4000, 0.1 M sodium citrate pH 5.3 and 0.1 M MgCl2. Crystals appeared within two to three days and reached maximum dimensions of 0.3×0.05×0.05 mm3 in approximately one week. The crystals were cryoprotected in the mother liquor plus the addition of 15% glycerol and flash frozen in a nitrogen stream at −180°C. X-ray intensity data were collected on beamline 8.3.1 at the Advanced Light Source and data were processed using MOSFLM as implemented in the CCP4 suite of programs (CCP4, 2004) (). The crystals took the spacegroup P1 with cell dimensions a = 29.4 Å, b = 32.2 Å, c = 50.6 Å, α = 106.1°, β = 91.6°, γ = 103.8°.The structure of the C10S Spx-αCTD complex was solved by molecular replacement using the oxidized Spx-αCTD structure as the search model (). Manual fitting and adjustment of the model resulted in the placement of amino acid residues 1–115 of Spx and residues 245–311 of αCTD into the electron density map using O . The initial protein model was subjected to rigid body refinement, followed by simulated annealing and positional and B-factor refinement using CNS . Simulated-annealing omit maps were calculated to ensure the correct placement of all residues and to avoid model bias. The final model included residues 1–115 of Spx, residues 245–311 of αCTD, and 40 solvent molecules. The final Rwork and Rfree are 23.6% and 27.8%, respectively, to 1.9 Å resolution. The stereochemistry of the final model was assessed with PROCHECK , which revealed 89.7% of all φ/ψ angles in the most favored regions of the Ramachandran plot and none in the disallowed regions. The coordinates and structure factors have been deposited in the RCSB with accession number 3IHQ. […]

Pipeline specifications

Software tools CNS, PROCHECK
Application Protein structure analysis
Organisms Bacillus subtilis