HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wu, J. Articles by Newton, A. Search for Related Content PubMed PubMed Citation Articles by Wu, J. Articles by Newton, A. Social Bookmarking                      What's this?

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.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				M. A. Ramirez-Romero, I. Masulis, M. A. Cevallos, V. Gonzalez, and G. Davila The Rhizobium etli {sigma}70 (SigA) factor recognizes a lax consensus promoter Nucleic Acids Res., March 9, 2006; 34(5): 1470 - 1480. [Abstract] [Full Text] [PDF]

				C. L. Richard, A. Tandon, and R. G. Kranz Rhodobacter capsulatus nifA1 Promoter: High-GC -10 Regions in High-GC Bacteria and the Basis for Their Transcription J. Bacteriol., February 1, 2004; 186(3): 740 - 749. [Abstract] [Full Text] [PDF]

				J. Wu, N. Ohta, J.-L. Zhao, and A. Newton A novel bacterial tyrosine kinase essential for cell division and differentiation PNAS, November 9, 1999; 96(23): 13068 - 13073. [Abstract] [Full Text] [PDF]

				J. Wu, N. Ohta, and A. Newton An essential, multicomponent signal transduction pathway required for cell cycle regulation in Caulobacter PNAS, February 17, 1998; 95(4): 1443 - 1448. [Abstract] [Full Text] [PDF]

				I. ROMBEL, A. NORTH, I. HWANG, C. WYMAN, and S. KUSTU The Bacterial Enhancer-binding Protein NtrC as a Molecular Machine Cold Spring Harb Symp Quant Biol, January 1, 1998; 63(0): 157 - 166. [Abstract] [PDF]