High-resolution characterization of DNA/protein complexes in living bacteria

Nicole A. Becker, Justin P. Peters, L James Maher III

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

The occurrence of DNA looping is ubiquitous. This process plays a well-documented role in the regulation of prokaryotic gene expression, such as the Escherichia coli lactose (lac) operon. Here, we present two complementary methods for high-resolution in vivo detection of DNA/protein binding within the bacterial nucleoid by using either chromatin immunoprecipitation combined with phage λ exonuclease digestion (ChIP-exo) or chromatin endogenous cleavage (ChEC), coupled with ligation-mediated polymerase chain reaction (LM-PCR) and Southern blot analysis. As an example we apply these in vivo protein-mapping methods to E. coli to show direct binding of architectural proteins in the Lac repressor-mediated DNA repression loop.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages95-115
Number of pages21
DOIs
StatePublished - Jan 1 2018

Publication series

NameMethods in Molecular Biology
Volume1837
ISSN (Print)1064-3745

Keywords

  • Architectural proteins
  • Chromatin endogenous cleavage (ChEC)
  • Chromatin immunoprecipitation (ChIP)
  • High-resolution mapping
  • Lac repression loop
  • Ligation-mediated PCR (LM-PCR)
  • Phage lambda exonuclease
  • Polymerase chain reaction (PCR)
  • Southern blot

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Fingerprint Dive into the research topics of 'High-resolution characterization of DNA/protein complexes in living bacteria'. Together they form a unique fingerprint.

  • Cite this

    Becker, N. A., Peters, J. P., & Maher III, L. J. (2018). High-resolution characterization of DNA/protein complexes in living bacteria. In Methods in Molecular Biology (pp. 95-115). (Methods in Molecular Biology; Vol. 1837). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-8675-0_6