| Assay Method Information | |
| | Inhibition Assay |
| Description: | Escherichia coli: Fluorescence-detected RNA polymerase assays with E.coli RNA polymerase were performed by a modification of the procedure of Khulman et al., 2004. Reaction mixtures contained (200): 0-100 nM test salt, 75nM E.coli RNA polymerase σ70 holoenzyme, 20nM 384 bp DNA fragment containing the bacteriophage T4 N25 promoter, 100 uM ATP, 100 uM GTP, 100 uM UTP, 100 uM CTP, 50mM Tris-HCl, pH 8.0, 100 nM KCl, 10mM MgCl2, 1mM DTT, 10uM/ml bovine serum albumin, and 5.5% glycerol. Reaction components other than DNA and NTPs were pre-incubated for 10 min at 37C. Reactions were carried out by addition of DNA and incubation for 5 min at 37C, followed by addition of NTPs and incubation for 60 min at 37C. DNA was removed by addition of 1ul 5mM CaCl2 and 2U DNasel, followed by incubation for 90 min at 37C. |
| Affinity data for this assay | |
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