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

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

Research output: Contribution to journalArticle

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Abstract

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
Volume272
Issue number34
DOIs
StatePublished - Aug 22 1997

Fingerprint

Caulobacter crescentus
Holoenzymes
DNA-Directed RNA Polymerases
Purification
Transcription
Genes
Escherichia coli
Sigma Factor
Single-Stranded DNA
Chromatography
Gene expression
Bacteria
Transcription Factors
Housekeeping
Cells
Cell Differentiation
Cell Cycle
Gene Expression
Proteins

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Purification, characterization, and reconstitution of DNA-dependent RNA polymerases from Caulobacter crescentus. / Wu, Jianguo; Ohta, Noriko; Benson, Andrew K.; Ninfa, Alexander J.; Newton, Austin.

In: Journal of Biological Chemistry, Vol. 272, No. 34, 22.08.1997, p. 21558-21564.

Research output: Contribution to journalArticle

Wu, Jianguo ; Ohta, Noriko ; Benson, Andrew K. ; Ninfa, Alexander J. ; Newton, Austin. / Purification, characterization, and reconstitution of DNA-dependent RNA polymerases from Caulobacter crescentus. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 34. pp. 21558-21564.
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