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