Figure 5. Functional role of Eco σ⁷⁰ W433 and Y394 in RPo formation.

(A) Simplified, two-step kinetic scheme for RPo formation (Roe et al., 1984; Buc and McClure, 1985) (R, RNAP; P, promoter; RP_i, intermediate complex). (B) Representative time trace of fluorescence increase (from Cy3 labelled promoter DNA) during RPo formation. The solid red line illustrates the non-linear regression fit to a single-exponential model (see ‘Materials and methods’), which described >90% of the fluorescence amplitude rise. (C) The RNAP-concentration dependence of the observed rate (k_obs) of RPo formation detected by Cy3 fluorescence (Ko and Heyduk, 2014) for Eco holoenzymes with σ⁷⁰ (wt) as well as σ⁷⁰ carrying substitutions W433A or Y394A. Error bars denote standard errors of the mean for ≥three independent measurements. (D) Summary of effects of σ⁷⁰ W433A and Y394A substitutions on thermodynamic and kinetic parameters of RPo formation. The data was normalized to the % observed with wild-type Eσ⁷⁰. (E) Equilibrium binding of ss nt-strand oligos of λ P_R promoter −10 element detected in the fluorescent RNAP beacon assay (Feklistov and Darst, 2011; Mekler et al., 2011) to Eco holoenzymes with σ⁷⁰, as well as σ⁷⁰ carrying substitutions W433A or Y394A.

DOI: http://dx.doi.org/10.7554/eLife.08504.015

Figure 5—figure supplement 1. RPo dissociation data.

(Left) Representative time trace of fluorescence decay after rapid mixing of pre-formed Eco RPo (with wild-type σ⁷⁰) into 1.1 M NaCl (Gries et al., 2010). The solid line illustrates the non-linear regression fit to a single-exponential model. (Right) Representative dissociation curves for holoenzymes containing wild-type σ⁷⁰ and W433A and Y394A substitutions.

DOI: http://dx.doi.org/10.7554/eLife.08504.016