Immobilization of Escherichia coli RNA Polymerase and Location of Binding Sites by Use of Chromatin Immunoprecipitation and Microarrays

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 ␤ subunit. The DNA was amplified and hybridized to "tiled" oligonucleotide microarrays representing the whole genome at 25-bp resolution. RNA polymerase (RNAP) in Escherichia coli is a key factor in gene expression and catalyzes the transcription of DNA to mRNA for all genes (reviewed in references 33 and 37). The core enzyme is composed of four subunits: two ␣, one ␤, one ␤Ј, and one . Core RNAP becomes transcriptionally active holoenzyme with the addition of a factor (Fig. 1A). E. coli possesses seven interchangeable factors, each with specificity for different promoters. Sigma factors function as global regulators of gene expression and mediate the transcriptional response to conditions under which a large number of genes need to be turned on or off, such as stationary phase or heat shock. Except for 54 , sigma factors do not bind to DNA except as part of the holoenzyme complex (9).To map promoters in bacteria, we sought a way to force RNAP to reside only at promoters so that identifying DNA fragments bound to RNAP in vivo would report promoter locations. A variety of small-molecule inhibitors of RNA polymerase were evaluated for the immobilization of RNAP, and rifampin was found to work best (M. Raffaelle, E. Kanin, J. Vogt, R. R. Burgess, A. Z. Ansari, unpublished data). The antibiotic rifampin inhibits bacterial growth by binding the ␤ subunit of RNAP just upstream of the active site, blocking the synthesis of RNAs longer than 2 to 3 nucleotides (nt) (11) (Fig. 1A). Rifampin has no effect on RNAP promoter binding to form closed complexes or on open complex formation (30) and has no effect on RNAP in vitro when added after elongating RNAP has cleared the promoter (12, 38). Upon addition of rifampin to a growing culture, the rifampin stops growth without killing cells by diffusion across bacterial membranes and tight binding to RNAP not engaged in transcription. RNAP holoenzyme complexed with rifampin can still bind to promoters but is trapped in an abortive cycle and unable to extend RNAs beyond 2 to 3 nt (Fig. 1A). However, RNAP molecules in elongation complexes with RNA and DNA are resistant to rifampin binding. Once elongating RNAPs terminate transcription and release RNA and DNA, they become susceptible to rifampin binding, which traps them in newly formed open complexes.A promoter is a DNA sequence to which RNAP binds and initiates the transcription of RNA. Knowledge of promoter locations is the first step in the elucidation of the transcriptional regulatory network. It allows the identification of which genes are cotranscribed and, from the DNA sequence, identification of regulatory motifs associated with different regulators. It also provides a basis for the interpretation of the bindin...

Genome-scale reconstruction of the Lrp regulatory network in Escherichia coli

“…The differential RNAP-binding levels are defined as the difference between the sums of RNAP-binding levels across all probes in a targeted gene's ORF under the 2 conditions. In the previous report (14), there was very little correlation between the log 2 ratio of RNAP-binding peaks obtained from the rifampicintreated cells and the expression of the nearest downstream ORF. However, we observed that the changes in RNAP-binding levels are correlated with the changes in the mRNA transcript levels in response to the exogenous leucine (Fig.…”

“…To determine the genome-wide Lrp-binding regions, we next performed a hybridization of the IP-DNA (Cy5 channel) and mock IP-DNA (Cy3 channel) onto the high-resolution whole-genome tiling microarrays, which contained a total of 371,034 oligonucleotides with 50-bp tiles overlapping every 25-bp on both forward and reverse strands (14). The normalized log 2 ratios obtained from the hybridization identify the genomic regions enriched in the IP-DNA sample compared with the mock IP-DNA sample and thereby represent a genome-wide map of in vivo interactions between Lrp protein and E. coli genome (Fig.…”

“…To verify the enrichment of the Lrp-binding regions in the DNA samples, 1 L of IP or mock-IP DNA was used to perform gene-specific real-time qPCR with the specific primers to the promoter regions. The IP and mock-IP DNA were then amplified by random DNA amplification method (14). Then, the amplified DNA samples were labeled and hybridized onto wholegenome-tiled microarrays (NimbleGen).…”

DNA microarray technologies for measuring protein–DNA interactions

“…NimbleGen 60-mer arrays tiling over 36 Mb of the Drosophila genome at 100 bp resolution also have been used [39]. Binding by Escherichia coli TFs has been examined using off-the-shelf Affymetrix E. coli antisense arrays [40], while NimbleGen tiling arrays have been used to map E. coli RNA Pol binding sites [41].…”

Expression levels of transcription factors in Escherichia coli: growth phase- and growth condition-dependent variation of 90 regulators from six families

“…Extensive efforts have recently been devoted to identifying the regulation target genes under the control of each TF by using highthroughput experimental systems such as the transcriptome and RNA-seq analyses of genome transcription patterns of TF-defective E. coli mutants (Cho et al, 2009;Raghavan et al, 2011;Ginnoukos et al, 2012) and upon exposure to stressful conditions such as changes in nutrients (Oh et al, 2002), exposure to heat shock (Richmond et al, 1999) or cold shock (Phadtare & Inouye, 2004), the presence of hydrogen peroxide (Zheng et al, 2001) or external metals (Lee et al, 2005), anaerobic conditions (Overton et al, 2006) and within biofilm (Ren et al, 2004). Direct measurement of TF-associated sites in vivo along the E. coli genome has been performed by ChIP-chip analysis (Herring et al, 2005;Grainger & Busby, 2008;Mooney et al, 2009). These in vivo analyses together provide information regarding TF-associated sites on the E. coli genome under the culture conditions employed, but are unable to identify the whole set of TF recognition sites because the functional forms of TFs are not always present in E. coli under laboratory culture conditions (see below) and because the TF-binding sites are often masked by other DNA-binding proteins.…”