Rapid RNA polymerase genetics: One-day, no-column preparation of reconstituted recombinant Escherichia coli RNA polymerase

Hong Tang, Konstantin Severinov, Alex Goldfarb, Richard H. Ebright

Research output: Contribution to journalArticlepeer-review

110 Citations (Scopus)

Abstract

We present a simple, rapid procedure for reconstitution of Escherichia coli RNA polymerase holoenzyme (RNAP) from individual recombinant α, β, β', and σ70 subunits. Hexahistidine-tagged recombinant α subunit purified by batch-mode metal-ion-affinity chromatography is incubated with crude recombinant β, β', and σ70 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, α-DNA interaction, catabolite gene activator protein (CAP)-independent transcription, and CAP-dependent transcription. Experiments with α(1-235), an α subunit C-terminal deletion mutant, establish that the procedure is suitable for biochemical screening of subunit lethal mutants.

Original languageEnglish
Pages (from-to)4902-4906
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume92
Issue number11
DOIs
Publication statusPublished - 23 May 1995
Externally publishedYes

Keywords

  • α subunit C-terminal deletion mutant
  • hexahistidine tag
  • metal ion-affinity chromatography
  • transcription
  • transcription activation

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