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 Esigma54, Esigma32, and Esigma73 were reconstituted exclusively from purified C. crescentus core and sigma factors. Reconstituted Esigma54 initiated transcription from the sigma54-dependent fljK promoter of C. crescentus in the presence of the transcription activator FlbD, and active Esigma32 specifically initiated transcription from the sigma32-dependent promoter of the C. crescentus heat-shock gene dnaK. For reconstitution of the Esigma73 holoenzyme, we overexpressed the C. crescentus rpoD gene in Escherichia coli and purified the full-length sigma73 protein. The reconstituted Esigma73 recognized the sigma70-dependent promoters of the E. coli lacUV5 and neo genes, as well as the sigma73-dependent housekeeping promoters of the C. crescentus pleC and rsaA genes. The ability of the C. crescentus Esigma73 RNA polymerase to recognize E. coli sigma70-dependent promoters is consistent with relaxed promoter specificity of this holoenzyme previously observed in vivo.
Highlights
Bacterial RNA polymerases (RNAP)1 are multi-subunit enzyme complexes that can be purified as the core polymerase (E) and the holoenzyme (E; reviewed in Refs. 4 and 5)
Biochemical studies of transcription in vitro have employed either the E54 holoenzyme reconstituted from purified Escherichia coli components [12, 13], the heterologous E54 holoenzyme reconstituted from the C. crescentus 54 and the E. coli core RNAP [14], or a partially purified C. crescentus RNAP [15]
Purification of RNA Polymerases—Cellular RNAP from C. crescentus was purified by fractionation of cell extracts with Polymin P, ammonium sulfate precipitation, and chromatography on heparin-agarose (Fig. 1A) and DEAE-cellulose (Fig. 1B), which removed many of the contaminating proteins
Summary
Bacterial RNA polymerases (RNAP) are multi-subunit enzyme complexes that can be purified as the core polymerase (E) and the holoenzyme (E; reviewed in Refs. 4 and 5). Bacterial RNA polymerases (RNAP) are multi-subunit enzyme complexes that can be purified as the core polymerase (E) and the holoenzyme The core RNAP, composed of the ␣2, , and Ј subunits, carries out RNA chain elongation, whereas the holoenzyme, which contains the sigma subunit (), recognizes specific promoter sequences. The 54 factor and transcription activator FlbD, which are encoded by Class II genes rpoN and flbD, are required for transcription of the Class III and IV genes (reviewed in Ref. 9). Sigma factors 54 [14] and 32 [16] have been overexpressed in E. coli and purified, the lack of a purified core RNAP has prevented the reconstitution of a transcription system exclusively from purified C. crescentus components. The availability of a defined, reconstituted transcription system will allow detailed analysis of the roles of RNAP and accessory factors in the transcriptional regulation of developmental genes during cell differentiation and division in this bacterium
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
