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Abstract
The promoter selectivity of Escherichia coli RNA polymerase (RNAP) is determined by the sigma subunit. The model prokaryote Escherichia coli K-12 contains seven species of the sigma subunit, each recognizing a specific set of promoters. For identification of the “constitutive promoters” that are recognized by each RNAP holoenzyme alone in the absence of other supporting factors, we have performed the genomic SELEX screening in vitro for their binding sites along the E. coli K-12 W3110 genome using each of the reconstituted RNAP holoenzymes and a collection of genome DNA segments of E. coli K-12. The whole set of constitutive promoters for each RNAP holoenzyme was then estimated based on the location of RNAP-binding sites. The first successful screening of the constitutive promoters was achieved for RpoD (σ70), the principal sigma for transcription of growth-related genes. As an extension, we performed in this study the screening of constitutive promoters for four minor sigma subunits, stationary-phase specific RpoS (σ38), heat-shock specific RpoH (σ32), flagellar-chemotaxis specific RpoF (σ28) and extra-cytoplasmic stress-response RpoE (σ24). The total number of constitutive promoters were: 129~179 for RpoS; 101~142 for RpoH; 34~41 for RpoF; and 77~106 for RpoE. The list of constitutive promoters were compared with that of known promoters identified in vivo under various conditions and using varieties of E. coli strains, altogether allowing the estimation of “inducible promoters” in the presence of additional supporting factors.
Highlights
The genome of Escherichia coli K-12, the most well-characterized model prokaryote, contains a total of more than 4,500 genes, which are transcribed by a single species of the RNA polymerase (RNAP)
As described in the previous report [27], we identified a total of 2,071 sites on the E. coli K-12 genome of binding of RNAP holoenzyme containing RpoD (σ70), the major sigma for transcription of most of the growth-related genes, and mapped the location of “constitutive promoters” that are recognized by RpoD holoenzyme alone in the absence of other DNAbinding proteins [Note that the “constitutive promoter” is defined as the promoter that is recognized by RNAP alone in the absence of supporting factors while the promoters that are detected only in vivo are defined as the “inducible promoters”, supposedly under the support of accessory regulatory factors]
In order to identify the whole set of constitutive promoters on the entire genome of E. coli K-12 W3110, we performed a mass-screening in vitro of the whole set of sequences that are recognized by the reconstituted holoenzymes, each containing only one specific minor sigma factor
Summary
The genome of Escherichia coli K-12, the most well-characterized model prokaryote, contains a total of more than 4,500 genes, which are transcribed by a single species of the RNA polymerase (RNAP). The intracellular concentration of RNAP is, approximately 2,000 molecules per genome, which is less than the total number of genes or operons [1,2,3]. The pattern of genome expression is determined by the selective distribution of a limited number of RNAP within the genome [4,5].
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