Escherichia coliσ70 and NusA proteins: I. Binding interactions with core RNA polymerase in solution and within the transcription complex

Abstract

This paper describes the binding interactions of Escherichia coli transcription factors σ⁷⁰ and NusA with core RNA polymerase, both free in solution and as a part of the functional transcription complex. High pressure liquid chromatography gel filtration and fluorescence techniques have been used to monitor the binding of these factors to core polymerase in solution at salt concentrations roughly comparable to the in vivo environment (250 mm-KCL. 50 mm-potassium phosphate (pH 7.5)); under these conditions all the interacting species exist separately as protein monomers. We find that σ⁷⁰ and NusA bind competitively to core polymerase with a 1:1 binding stoichiometry in this milieu, and that NusA does not bind to the polymerase holoenzyme. Association constants of approximately 2 × 10⁹ and 1 × 10⁷m⁻¹ have been measured for the σ⁷⁰-core polymerase interaction and for the NusA-core polymerase interaction, respectively. These findings are consistent with the original formulation of the NusA-σ⁷⁰ cycle put forward by Greenblatt & Li, and provide the basis for a further (and preliminary) quantitative examination of these same interactions within the transcription complex. We use a number of molecular biological techniques, together with data from the literature, to estimate these binding constants in various phases of the transcription cycle. In keeping with our results in solution, we find that the effective binding affinity of σ⁷⁰ for core polymerase within the “open” promoter-polymerase complex is at least 500-fold greater than that of NusA. As the transcription complex moves from the initiation to the elongation phase these relative binding affinities are reversed; the average association constant of NusA for the core polymerase in the elongation complex remains practically the same as in free solution (approx. 3 × 10⁷m¹), while the affinity of σ⁷⁰ for core polymerase in this complex drops to less than 5 × 10⁵m¹. These results are used to begin to define the basic conformational states and interaction potentials of core polymerase in the various stages of the transcription cycle.