Competition among seven Escherichia coli sigma subunits: relative binding affinities to the core RNA polymerase

Abstract

Seven different species of the RNA polymerase sigma subunit exist in Escherichia coli, each binding to a single species of the core enzyme and thereby directing transcription of a specific set of genes. To test the sigma competition model in the global regulation of gene transcription, all seven E.coli sigma subunits have been purified and compared for their binding affinities to the same core RNA polymerase (E). In the presence of a fixed amount of sigma(70), the principal sigma for growth-related genes, the level of Esigma(70) holoenzyme formation increased linearly with the increase in core enzyme level, giving an apparent K:(d) for the core enzyme of 0.26 nM. Mixed reconstitution experiments in the presence of a fixed amount of core enzyme and increasing amounts of an equimolar mixture of all seven sigma subunits indicated that sigma(70) is strongest in terms of core enzyme binding, followed by sigma(N), sigma(F), sigma(E)/sigma(FecI), sigma(H) and sigma(S) in decreasing order. The orders of core binding affinity between sigma(70) and sigma(N) and between sigma(70) and sigma(H) were confirmed by measuring the replacement of one core-associated sigma by another sigma subunit. Taken together with the intracellular sigma levels, we tried to estimate the number of each holoenzyme form in growing E. coli cells.

Figure 1

Determination of the affinity between the σ⁷⁰ subunit and the core enzyme. (A) Core enzyme saturation. A fixed amount (0.4 nM) of the σ⁷⁰ subunit and increasing amounts of the core enzyme were mixed and the holoenzyme (Eσ⁷⁰) formed was isolated by Superdex-200 chromatography using a SMART system. The amount of core enzyme-bound σ⁷⁰ was measured by SDS–PAGE followed by staining with SYPRO Orange. The K_d was estimated using Sigma Plot Software. (B) σ⁷⁰ saturation. A fixed amount (20 pmol/50 µl or 400 nM) of the core enzyme was mixed with increasing amounts of the σ⁷⁰ subunit and the amount of Eσ⁷⁰ holoenzyme was measured as in (A).

Figure 2

Purification of various σ subunits and fractionation of core enzyme-bound and unbound σ subunits. (A) SDS–PAGE of purified σ subunits. Seven species of the σ subunit were purified as described in Materials and Methods and analyzed by SDS–PAGE (9% gel for lanes 1–5, 15% gel for lanes 6–9). Gels were stained with Coomassie brilliant blue. M_r markers used were rabbit muscle phosphorylase (97.4 kDa), bovine serum albumin (66.2 kDa), hen egg white ovalbumin (45 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa) and hen egg white lysozyme (14.4 kDa). (B) Mixtures of seven σ subunits and the core enzyme were fractionated by gel filtration through a Superdex 200 column (0.25 × 30 cm) with a SMART system (Pharmacia, Sweden). The elution positions of holoenzymes and unbound σ subunits are indicated by arrows.

Figure 3

Mixed reconstitution of various holoenzymes. A fixed amount (20 pmol) of the core enzyme was mixed with increasing amounts of an equimolar mixture of seven σ subunits (σ⁷⁰, σ^S, σ^N, σ^F, σ^H, σ^E and σ^FecI) and, after incubation for 10 min at 30°C, fractionated at 20°C by gel filtration chromatography through a Superdex 200 column (0.25 × 30 cm) using a SMART system as shown in Figure 2B. The molar ratio of combined σ subunits to the core enzyme was 1.75 (A), 7.0 (B) and 14 (C). The amounts of core enzyme-bound σ subunits were determined as described in Figure 1.

Figure 4

Replacement of the core enzyme-bound σ subunit. An equimolar mixture of the core enzyme (20 pmol) and the first σ subunit (20 pmol) was incubated for 10 min at 30°C and then 20 pmol of the second σ subunit was added. After incubation for various times as indicated, the core enzyme-bound σ subunits were measured as in Figure 1. The combined amount of core enzyme-bound σ subunits was the same as the core enzyme within the sensitivity range of protein determination. The first and second σ subunits were (A) σ⁷⁰ and σ^H, (B) σ^H and σ⁷⁰, (C) σ⁷⁰ and σ^N and (D) σ^N and σ⁷⁰.