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

Fingerprint

Lac Repressors
Escherichia coli
Bacteria
Exonucleases
Lac Operon
Chromatin Immunoprecipitation
DNA
DNA-Binding Proteins
Gene Expression Regulation
Southern Blotting
Bacteriophages
Chromatin
Ligation
Digestion
Carrier Proteins
Proteins
Polymerase Chain Reaction

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

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

High-resolution characterization of DNA/protein complexes in living bacteria. / Becker, Nicole A.; Peters, Justin P.; Maher III, L James.

Methods in Molecular Biology. Humana Press Inc., 2018. p. 95-115 (Methods in Molecular Biology; Vol. 1837).

Research output: Chapter in Book/Report/Conference proceedingChapter

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