Rapid RNA polymerase genetics: one-day, no-column preparation of reconstituted recombinant Escherichia coli RNA polymerase.
收藏PubMed Central1995-05-23 更新2026-05-02 收录
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https://pmc.ncbi.nlm.nih.gov/articles/PMC41815/
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资源简介：
We present a simple, rapid procedure for reconstitution of Escherichia coli RNA polymerase holoenzyme (RNAP) from individual recombinant alpha, beta, beta', and sigma 70 subunits. Hexahistidine-tagged recombinant alpha subunit purified by batch-mode metal-ion-affinity chromatography is incubated with crude recombinant beta, beta', and sigma 70 subunits from inclusion bodies, and the resulting reconstituted recombinant RNAP is purified by batch-mode metal-ion-affinity chromatography. RNAP prepared by this procedure is indistinguishable from RNAP prepared by conventional methods with respect to subunit stoichiometry, alpha-DNA interaction, catabolite gene activator protein (CAP)-independent transcription, and CAP-dependent transcription. Experiments with alpha (1-235), an alpha subunit C-terminal deletion mutant, establish that the procedure is suitable for biochemical screening of subunit lethal mutants. IMAGES:
提供机构：
National Academy of Sciences
创建时间：
1995-05-23
