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Journal of Bacteriology, September 2005, p. 6166-6174, Vol. 187, No. 17
0021-9193/05/$08.00+0     doi:10.1128/JB.187.17.6166-6174.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Immobilization of Escherichia coli RNA Polymerase and Location of Binding Sites by Use of Chromatin Immunoprecipitation and Microarrays{dagger}

Christopher D. Herring,1,2,4 Marni Raffaelle,2 Timothy E. Allen,1 Elenita I. Kanin,2 Robert Landick,4 Aseem Z. Ansari,2,3* and Bernhard Ø. Palsson1*

Department of Bioengineering, University of California San Diego, San Diego, California,1 Department of Biochemistry, University of Wisconsin Madison, Madison, Wisconsin,2 Genome Center of Wisconsin, Madison, Wisconsin,3 Department of Bacteriology, University of Wisconsin Madison, Madison, Wisconsin4

Received 20 April 2005/ Accepted 14 June 2005

The genome-wide location of RNA polymerase binding sites was determined in Escherichia coli using chromatin immunoprecipitation and microarrays (chIP-chip). Cross-linked chromatin was isolated in triplicate from rifampin-treated cells, and DNA bound to RNA polymerase was precipitated with an antibody specific for the ß' subunit. The DNA was amplified and hybridized to "tiled" oligonucleotide microarrays representing the whole genome at 25-bp resolution. A total of 1,139 binding sites were detected and evaluated by comparison to gene expression data from identical conditions and to 961 promoters previously identified by established methods. Of the detected binding sites, 418 were located within 1,000 bp of a known promoter, leaving 721 previously unknown RNA polymerase binding sites. Within 200 bp, we were able to detect 51% (189/368) of the known {sigma}70-specific promoters occurring upstream of an expressed open reading frame and 74% (273/368) within 1,000 bp. Conversely, many known promoters were not detected by chIP-chip, leading to an estimated 26% negative-detection rate. Most of the detected binding sites could be associated with expressed transcription units, but 299 binding sites occurred near inactive transcription units. This map of RNA polymerase binding sites represents a foundation for studies of transcription factors in E. coli and an important evaluation of the chIP-chip technique.

* Corresponding author. Mailing address for Bernhard Ø. Palsson: UC San Diego Bioengineering, 9500 Gilman Drive, Dept 0412, La Jolla, CA 92093-0412. Phone: (858) 534-5668. Fax: (858) 822-3120. E-mail: bpalsson{at}ucsd.edu. Mailing address for Aseem Z. Ansari: Department of Biochemistry, 433 Babcock Drive, University of Wisconsin-Madison, Madison, WI 53706. Phone: (608) 265-4690. Fax: (608) 262-3453. E-mail: ansari{at}biochem.wisc.edu.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, September 2005, p. 6166-6174, Vol. 187, No. 17
0021-9193/05/$08.00+0     doi:10.1128/JB.187.17.6166-6174.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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