New improved methods were developed for the purification to apparent homogeneity of α,β,β', and σ subunits of Escherichia coli RNA polymerase (RNAP) from corresponding overproducing strains. The purified subunits were assembled into enzymatically active RNAP holoenzyme (α2ββ'σ) using the optimal subunit molar ratio (α:β:β':σ = 2:8:4:1) at a total protein concentration of 0.5 mg/ml. The presence of σ subunit and 10 μM ZnCl2 in the reconstitution mixture increased the yield of RNAP ∼ 4 times. The assembled RNA polymerase was purified by two successive chromatographic steps using size-exclusion Superose 6 and anion exchange Mono Q FPLC columns, which resulted in the electrophoretically homogeneous holoenzyme with overall yield of 56%. The specific activity of the recombinant RNAP estimated by the standard T4 transcription assay was 6.5 nmol of [3H]UTP incorporated into acid-insoluble RNA product per microgram of RNAP per 1 h.
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