Reconstitution of RNA polymerase from deep-sea piezophilic Shewanella violacea

概要

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RNA polymerase from cells of the deep-sea bacterium Shewanella violacea DSS12 was purified using three chromatographic steps. An in vitro transcription assay revealed that the purified enzyme was σ^<70> containing RNA polymerase. The enzyme activity was inhibited in the presence of rifampicin when the sensitive domain was targeted. The rpoA, rpoB, rpoC, rpoZ and rpoD genes encoding for the α, β, β', ω and σ subunits of RNA polymerase were cloned into expression plasmids, designated as pQSVA pQSVB pQSVZ and pQSVD, to overproduce these proteins were constructed and the proteins were purified using a Ni^<2+> affinity column. In vitro reconstitution using all proteins for the holoenzyme (α, β, β' and σ^<70>) was carried out and the activity of the recombinant RNA polymerase was detected.
近畿大学の論文
2004-12-20