Protein kinase a

The recombinant gpT5.026 protein was labeled with an isotope ³²P using the catalytic subunit of protein kinase A (Sigma), as described [20 (link),21 (link),22 (link)], followed by a clean-up on Ni–NTA (Qiagen). Far-Western blotting was performed as previously described [21 (link),23 (link)]. Briefly, 1 μg of purified proteins (gpT5.026 or gp2) were incubated with 20 units of protein kinase A in PKA buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 30 mM DTT, 10 mM MgCl₂) in the presence of 0.4 mCi of γ-[³²P] ATP at 30 °C for 1 h, followed by a clean-up on Ni-NTA (Qiagen). 1 μg of RNAP core enzymes or individual RNAP subunits (α, β, β’, and σ⁷⁰) were applied as small drops on a Hybond ECL membrane and annealed for 3 min at 50 °C. Then, membranes were blocked in PROB buffer (20 mM 1,4-Piperazinediethanesulfonic acid (PIPES) (pH 7.4), 200 mM KCl, 1 mM DTT, 2 mM MgCl₂, 10% glycerol, 0.5% Tween-20, 1% nonfat dried milk) for 2 h at room temperature. Afterwards, each piece of membrane was enveloped in a parafilm sack containing 150 μL of radiolabeled proteins solutions with/without added non-labeled proteins (depending on the design of experiment) in PROB buffer and incubated for 2 h at room temperature. Next, membranes were washed three times with 1 mL of PROB buffer and dried at room temperature for 15 min. Results were revealed using a PhosphorImager (Molecular Dynamics).