High-Resolution Characterization of DNA/Protein Complexes in Living Bacteria

Abstract

The occurrence of DNA looping is ubiquitous. This process plays a well-documented role in the regulation of prokaryotic gene expression, such as in regulation of 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.

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.