Studies of the distribution of Escherichia coli cAMP-receptor protein and RNA polymerase along the E. coli chromosome

Abstract

Chromatin immunoprecipitation and high-density microarrays have been used to monitor the distribution of the global transcription regulator Escherichia coli cAMP-receptor protein (CRP) and RNA polymerase along the E. coli chromosome. Our results identify targets occupied by CRP and genes transcribed by RNA polymerase in vivo. Many of the loci of CRP binding are at known CRP regulated promoters. However, our results show that CRP also interacts with thousands of weaker sites across the whole chromosome and that this "background" binding can be used as a probe for organization within the E. coli folded chromosome. In rapidly growing cells, we show that the major sites of RNA polymerase binding are approximately 90 transcription units that include genes needed for protein synthesis. Upon the addition of rifampicin, RNA polymerase is distributed among >500 functional promoters. We show that the chromatin immunoprecipitation and high-density-microarrays methodology can be used to study the redistribution of RNA polymerase induced by environmental stress, revealing previously uncharacterized aspects of RNA polymerase behavior and providing an alternative to the "transcriptomics" approach for studying global transcription patterns.

Fig. 1.

Genome-wide distribution of MelR and CRP in E. coli. (A) Results of a ChIP-chip experiment to analyze the sequence distribution of DNA fragments precipitated with anti-MelR antibodies. The DNA, obtained from immunoprecipitation of MG1655 nucleoprotein by anti-MelR, was labeled with Cy5 and compared with a Cy3-labeled control from the ΔmelR strain. The data are plotted as Cy5/Cy3 ratios (y axis), displayed as a function of the location of the probe on the MG1655 chromosome (x axis). (Inset) An expansion of the mel-operon-regulatory region. (B) Results of a ChIP-chip experiment to analyze the sequence distribution of DNA fragments precipitated with anti-CRP antibodies. The DNA, obtained from immunoprecipitation of MG1655 nucleoprotein by anti-CRP, was labeled with Cy5 and compared with a Cy3-labeled control from the Δcrp strain. The data are plotted as Cy5/Cy3 ratios (y axis), displayed as a function of the location of the probe on the MG1655 chromosome (x axis). The locations of selected signals are labeled. The complete data set can be accessed at www.ogt.co.uk/ogt_services/chipzone.htm, and Table 1 lists details of the targets corresponding to the principal signals. (C) Expansion of selected regulatory regions highlighted in B. (D) Low-resolution plot of ChIP-chip data from immunoprecipitations, with anti-CRP, of nucleoprotein from cells grown without (black) or with glucose (dark gray). Cy5/Cy3 ratios were calculated as an average over a 200,000-bp moving window and are plotted as a function of location on the MG1655 chromosome (x axis). For comparison, data from the MelR experiment in A are included (light gray).

Fig. 2.

Genome-wide distribution of RNA polymerase in E. coli. (A) Results of a ChIP-chip experiment to analyze the sequence distribution of DNA fragments precipitated with anti-RNA-polymerase β-subunit antibodies. The DNA, obtained from immunoprecipitation of nucleoprotein from rapidly growing MG1655 cells, was labeled with Cy5 and compared with a Cy3-labeled control. The data are plotted as Cy5/Cy3 ratios (y axis), displayed as a function of the location of the probe on the MG1655 chromosome (x axis). The locations of selected signals are labeled. The complete data set can be accessed at www.ogt.co.uk/ogt_services/chipzone.htm, and Table 2 lists details of the top 20 targets. (B) As in A, but cells were treated with rifampicin before immunoprecipitation. The complete data set is available online (see above) and is presented as a file that can be downloaded into a genome browser. Table 3 lists details of the 529 targets identified in this experiment. (C) Expansion of selected regions from data in B.

Fig. 3.

Redistribution of RNA polymerase in E. coli. (A) Results of a ChIP-chip experiment to analyze the redistribution of RNA polymerase in cells after the addition of IPTG. The DNA, obtained from immunoprecipitation of nucleoprotein from MG1655 cells growing in the presence or absence of IPTG, was labeled with Cy5 or Cy3, respectively. The data are plotted as Cy5/Cy3 ratios (y axis), displayed as a function of the location of the probe on the MG1655 chromosome (x axis). (Inset) An expansion of the data for the lac operon. (B) As in A, but cells were treated with salicylic acid instead of IPTG. The locations of selected up- and down-regulated signals are labeled. The complete data set can be accessed at www.ogt.co.uk/ogt_services/chipzone.htm and is presented as a file that can be downloaded into a genome browser. (C) Expansion of selected regions from data in B. Data from experiments with both antibodies directed against the RNA polymerase β and σ⁷⁰ subunits are shown (complete data set for σ⁷⁰ binding available online).