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Volume 272, Number 34, Issue of August 22, 1997 pp. 21558-21564
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Purification, Characterization, and Reconstitution of DNA-dependent RNA Polymerases from Caulobacter crescentus

(Received for publication, March 17, 1997, and in revised form, June 4, 1997)

From the Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544

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 E⁵⁴, E³², and E⁷³ were reconstituted exclusively from purified C. crescentus core and sigma factors. Reconstituted E⁵⁴ initiated transcription from the ⁵⁴-dependent fljK promoter of C. crescentus in the presence of the transcription activator FlbD, and active E³² specifically initiated transcription from the ³²-dependent promoter of the C. crescentus heat-shock gene dnaK. For reconstitution of the E⁷³ holoenzyme, we overexpressed the C. crescentus rpoD gene in Escherichia coli and purified the full-length ⁷³ protein. The reconstituted E⁷³ recognized the ⁷⁰-dependent promoters of the E. coli lacUV5 and neo genes, as well as the ⁷³-dependent housekeeping promoters of the C. crescentus pleC and rsaA genes. The ability of the C. crescentus E⁷³ RNA polymerase to recognize E. coli ⁷⁰-dependent promoters is consistent with relaxed promoter specificity of this holoenzyme previously observed in vivo.

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