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

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.