Purification, characterization, and reconstitution of DNA-dependent RNA polymerases from Caulobacter crescentus

Jianguo Wu, Noriko Ohta, Andrew K. Benson, Alexander J. Ninfa, Austin Newton

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8 Scopus citations


Cell differentiation in the Caulobacter crescentus cell cycle requires differential gene expression that is regulated primarily at the transcriptional level. Until now, however, a defined in vitro transcription system for the biochemical study of developmentally regulated transcription factors had not been available in this bacterium. We report here the purification of C. crescentus RNA polymerase holoenzymes and resolution of the core RNA polymerase from holoenzymes by chromatography on single- stranded DNA cellulose. The three RNA polymerase holoenzymes Eσ54, Eσ32, and Eσ73 were reconstituted exclusively from purified C. crescentus core and sigma factors. Reconstituted Eσ54 initiated transcription from the σ54-dependent fljK promoter of C. crescentus in the presence of the transcription activator F1bD, and active Eσ32 specifically initiated transcription from the σ32-dependent promoter of the C. crescentus heatshock gene dnaK. For reconstitution of the Eσ73 holoenzyme, we overexpressed the C. crescentus rpoD gene in Escherichia coli and purified the full-length σ73 protein. The reconstituted Eσ73 recognized the σ70-dependent promoters of the E. coli lacUV5 and neo genes, as well as the σ73-dependent housekeeping promoters of the C. crescentus pleC and rsaA genes. The ability of the C. crescentus Eσ73 RNA polymerase to recognize E. coli σ70-dependent promoters is consistent with relaxed promoter specificity of this holoenzyme previously observed in vivo.

Original languageEnglish (US)
Pages (from-to)21558-21564
Number of pages7
JournalJournal of Biological Chemistry
Issue number34
StatePublished - Aug 22 1997

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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