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. 2017 Jun 30;12(6):e0179181.
doi: 10.1371/journal.pone.0179181. eCollection 2017.

The whole set of the constitutive promoters recognized by four minor sigma subunits of Escherichia coli RNA polymerase

Tomohiro Shimada  1   2 , Kan Tanaka  2 , Akira Ishihama  1
Affiliations

The whole set of the constitutive promoters recognized by four minor sigma subunits of Escherichia coli RNA polymerase

Tomohiro Shimada et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. SELEX-chip search for RNAP RpoS holoenzyme-binding sequences on the E. coli K-12 genome.
The y-axis represents the relative number of RpoS holoenzyme-bound DNA fragments whereas x-axis represents the position on the E. coli K-12 genome, in base pair. The adjacent gene on E. coli K-12 genome of peak position was indicated for high intensity peaks. The peaks located within spacer regions are shown in green color, while peaks located within open reading frames are shown in orange color. The list of RpoS holoenzyme-binding sites is described in Table 1.
Fig 2
Fig 2. SELEX-chip search for RNAP RpoH holoenzyme-binding sequences on the E. coli K-12 genome.
The y-axis represents the relative number of RpoH holoenzyme-bound DNA fragments whereas x-axis represents the position on the E.coli K-12 genome, in base pair. The adjacent gene on E. coli K-12 genome of peak position was indicated for high intensity peaks. The peaks located within spacer regions are shown in green color, while peaks located within open reading frames are shown in orange color. The list of RpoH holoenzyme-binding sites is described in Table 3.
Fig 3
Fig 3. SELEX-chip search for RNAP RpoF holoenzyme-binding sequences on the E. coli K-12 genome.
The y-axis represents the relative number of RpoF holoenzyme-bound DNA fragments whereas x-axis represents the position on the E. coli K-12 genome, in base pair. The adjacent gene on E. coli K-12 genome of peak position was indicated for high intensity peaks. The peaks located within spacer regions are shown in green color, while peaks located within open reading frames are shown in orange color. The list of RpoF holoenzyme-binding sites is described in Table 4.
Fig 4
Fig 4. SELEX-chip search for RNAP RpoE holoenzyme-binding sequences on the E. coli K-12 genome.
The y-axis represents the relative number of RpoE holoenzyme-bound DNA fragments whereas x-axis represents the position on the E.coli genome, in base pair. The adjacent gene on E. coli K-12 genome of peak position was indicated for high intensity peaks. The peaks located within spacer regions are shown in green color, while peaks located within open reading frames are shown in orange color. The list of RpoE holoenzyme-binding sites is described in Table 5.
Fig 5
Fig 5. Intracellular concentrations of seven sigma factors in E. coli K-12 W3110 type-A strain.
E. coli K-12 W3110 type-A strain was grown in LB medium at 37°C with shaking. Cells were harvested at various times and cell lysates were subjected to the quantitative immuno-blot analysis of all seven sigma factors as described in Materials and Methods. [A] The sigma levels at exponential growth phase; [B] the sigma levels in the stationary phase.
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Grants and funding

This work was supported by National Institute of Genetics to YY; MEXT Grants-in-Aid for Scientific Research (A) (21241047), (B) (18310133), and (C) (25430173) to AI; MEXT Grant-in-Aid for Young Scientists (B) (24710214) to TS; Research Fund from IFO (Institute for Fermentation, Osaka) to TS; funding from the MEXT Cooperative Research Program of Network Joint Research Center for Materials and Devices to AI and KT; and funding to AI and TS from the MEXT-Supported Program for the Strategic Research Foundation at Private Universities 208-2012 (S0801037) to Hosei University.