E coli rna polymerase holoenzyme

In vitro transcription assays on the promoter DNA of hrpX was performed. The promoter template DNA fragment of XAC1266 (P_hrpX) was generated by PCR amplification using XccWT genomic DNA as template and primers phrpX up and phrpX-ivt down (S1 Table). The product includes the promoter region and 200 pb downstream from the annotated transcription start site [12 (link)]. For in vitro transcription, 16 or 32 pmoles of HrpG or HrpG-R210C were incubated for 20 min at room temperature in transcription buffer (5 mM TrisHCl pH 7, 25 mM KCl, 2.5% glycerol, 2 mM EDTA, 10 mM MgCl₂, 1 mM DTT) containing 1 pmol of promoter template DNA. Then, 1 U of E. coli RNA polymerase Holoenzyme (sigma saturated) (Epicentre, Madison, WI, USA) was added and further incubate for 15 min at 28°C. Next, a NTP mixture (10 mM each of ATP, CTP and GTP and 50 mM [α-³²P] UTP (3000 Ci/mmol, 10 mCi/ml)) was added to start transcription. After incubation at 28°C for 20 min, reactions were stopped by addition of loading buffer (95% formamide, 10mM NaOH, 0.05% xylene cyanol, 0.05% bromophenol blue) and incubated at 80°C for 2 min. Transcription products were run on 6% denatured polyacrylamide gel containing 7 M urea in 0,5X TBE (89 mM Tris Base, 89 mM boric acid, 2 mM EDTA) electrophoresis buffer. The transcripts obtained were visualized by autoradiography. Experiments were repeated three times with similar results.

E. coli RNA polymerase holoenzyme harbouring σ⁷⁰ (RNAP) was obtained from Epicentre Technologies (Madison). The His₆-LexA protein was overexpressed and purified as described in [29 (link)] and stored in 20 mM Tris (pH 7.3), 200 mM NaCl at -80°C. The His₆-IscR protein was overexpressed, purified and its concentration determined as described in [11 (link)]. To induce the synthesis of AsnC protein, an overnight culture of E. coli BL21 (DE3)pLysE strain grown on an agar plate, containing ampicilin (100 μg ml^-1) and chloramphenicol (25 μg ml^-1), harbouring pAsnC was grown to an optical density at 600 nm (OD₆₀₀) of 0.6 when 0.8 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the culture. After 4 h of growth the cells were harvested and the N-terminally His₆-tagged AsnC was affinity purified by Ni-chelate chromatography (Quiagen) and stored at 4°C in 50 mM NaH₂PO₄ (pH 8), 300 mM NaCl, 250 mM imidazole. The concentrations of the LexA and AsnC proteins were determined using a NanoDrop 1000 (Thermo Scientific) using the extinction coefficients at 280 nm of 6990 M^-1 cm^-1 and of 10555 M^-1 cm^-1, respectively.