E. coli RNA Polymerase Core Enzyme and Sigma-Saturated Holoenzyme

E.coli RNA ポリメラーゼ Core Enzyme及びHoloenzyme はりファムピシリン-感受性株BL21から分離されました*。EPICENTRE社はsigma subunitを検出しない精製されたCore Enzymeと100% Sigma-Saturated (σ70)-Holoenzymeを提供できる唯一の会社です。

Core Enzymeは、sigma要素を欠いているのでバクテリアやバクテリオファージDNAのプロモーターで特定の転写を開始しませんので、転写開始のメカニズムを研究するのに役に立ちます。しかしながらCore Enzyme非特異的な転写産物を合成します。sigma-saturated Holoenzymeはプロモーターを含む様々なdouble-stranded DNA templatesを転写することにおいて非常に効率的です。

Figure 1. Subunit patterns of E. coli RNA Polymerase Core Enzyme and Holoenzyme preparations.

Equivalent amounts of each enzyme were separated by electrophoresis on an SDS 15% polyacrylamide gel, stained with Coomassie Blue, and dried. The sigma-70 subunit is 100% saturating in the Holoenzyme, but is absent in the Core Enzyme.

Unit Definition

One unit of E. coli RNA Polymerase catalyzes the incorporation of 1 nmole of a ribonucleoside triphosphate into RNA in 10 minutes at 37°C using standard assay conditions.

Storage Buffer

50% glycerol containing 50 mM Tris-HCl (pH7.5), 250 mM NaCl, 0.1 mM EDTA, and 1 mM DTT.

Quality Control

E. coli Core and Holoenzyme preparations are free of detectable DNase and RNase activities.

References

1. Burgess, R and Jendrisak, J (1975) Biochemistry 14, 4634.
2. Chamberlain, MJ et al. (1979) J. Biol. Chem. 254, 10061.

* Holoenzyme and Core Enzyme refer to E. coli RNA Polymerase that are, respectively, with or without the sigma-70 (σ70) subunit, as determined by SDS-PAGE, and by initiation of transcription from the host promoter on bacteriophage T7 DNA and lack thereof by Core RNA Polymerase. Contamination with other “minor” sigma factor(s) (such as σ35), transcription factors (such as GreA or GreB), or transcription accessory proteins is not excluded.

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