Immobilization of Escherichia coli RNA polymerase and location of binding sites by use of chromatin immunoprecipitation and microarrays.

TitleImmobilization of Escherichia coli RNA polymerase and location of binding sites by use of chromatin immunoprecipitation and microarrays.
Publication TypeJournal Article
Year of Publication2005
AuthorsHerring CD, Raffaelle M, Allen TE, Kanin EI, Landick R, Ansari AZ, Palsson BØ
JournalJournal of bacteriology
Volume187
Issue17
Pagination6166-74
PubMed Date2005 Sep
ISSN0021-9193
KeywordsBase Sequence, Binding Sites, Chromatin, DNA Primers, DNA-Directed RNA Polymerases, Enzymes, Immobilized, Escherichia coli, Gene Expression Regulation, Bacterial, Gene Expression Regulation, Enzymologic, Oligonucleotide Array Sequence Analysis, Rifampin, Transcription, Genetic
Abstract

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 beta' 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 sigma70-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.

Alternate JournalJ. Bacteriol.
PubMed ID16109958

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