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 Ϸ90 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.chromatin immunoprecipitation ͉ genomics ͉ stringent response ͉ transcription I n growing Escherichia coli, Ϸ2,000 RNA polymerase molecules are distributed unevenly among 4,300 genes, and Ͼ150 transcription factors play a key role in managing this distribution (1-3). It is important to understand the genome-wide profile of RNA polymerase binding and the role of each of the transcription factors. Here, we describe the use of chromatin immunoprecipitation (ChIP) and high-density microarrays (known as the ChIP-chip approach) to study the whole-genome DNA-binding properties of E. coli cAMP-receptor protein (CRP), one of the principal global transcription regulators in E. coli, and RNA polymerase.CRP is one of the seven ''global'' transcription factors in E. coli known to regulate Ͼ50% of the cell's transcription units (3). CRP's activity is triggered by binding of the second messenger cAMP in response to glucose starvation and other stresses (4). Many catalogues of CRP regulated promoters have been assembled, based on either transcriptome analysis (5-7) or bioinformatics (8, 9). These analyses conclude that CRP directly affects up to 200 promoters, although neither approach directly measures CRP binding at promoters. The computational analysis of Robison et al. (8) identified Ϸ200 high-affinity DNA targets for CRP, predominantly located in noncoding parts of the E. coli chromosome. That study also predicted Ͼ10,000 lower-affinity CRP targets scattered throughout the chromosome, with little bias to noncoding sequences. In this work, we used ChIP-chip analysis to take snapshots of the distribution of CRP in living E. coli cells. The advantage of this methodology (reviewed in ref. 10) is that it provides a direct measure of binding at different targets, enabling us to identify specific sites where CRP binds...

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

“…From the genome-wide mapping results, we were also able to show that: (i) A total of 138 Lrp-binding regions were identified, Ϸ84% of which were located within noncoding regions, whereas the remaining Ϸ16% were found within coding regions; (ii) 34 and 92 Lrp-binding regions were identified in exponentially growing cells in the presence and absence of exogenous leucine, respectively, indicating that Lrp bindings to the E. coli genome are dramatically sensitive to the addition of exogenous leucine; and (iii) the Lrp-binding sites on the E. coli genome under stationary growth condition (134 Lrp-binding regions identified) indicated that Lrp plays pivotal roles in the transcriptional regulation under stationary growth conditions. The high number of Lrp-binding regions was not surprising, given that global transcription factors such as Fnr, Crp, Ihf, Fis, and Hns specifically bind to between Ϸ63 and Ϸ224 target regions (22)(23)(24).…”

Circuitry Linking the Catabolite Repression and Csr Global Regulatory Systems of Escherichia coli

“…The cAMP-CRP complex mediates hierarchical utilization of nonpreferred carbon sources, referred to as carbon catabolite repression (CCR), by activating the expression of genes required for the transport and utilization of alternative carbon sources (49). cAMP-CRP also influences the expression of genes not directly involved in carbon metabolism such as those encoding ribosomal proteins, tRNAs, amino acid biosynthesis enzymes, heat shock proteins, sRNAs, and perhaps as many as 70 transcription factors (45)(46)(47)(50)(51)(52)(53)(54)(55). cAMP levels and cAMP-CRP regulatory functions have been suggested to respond to both the carbon status and the nitrogen status of the cell, leading to reorganization of the proteome (56).…”

Insights into transcriptional regulation and σ competition from an equilibrium model of RNA polymerase binding to DNA

“…In fact, discrepancies in these numbers have led to the common perceptions that (i) as most RNAP is elongating, only a minor fraction could be bound to DNA nonspecifically (27,28), and (ii) cellular levels of 70 are much less than that of RNAP (29); thus, s may compete because of their excess over free RNAP. We have remeasured the levels of E, 70 , 32 , and E in E. coli K12 MG1655.…”