Interaction of σ⁷⁰ with Escherichia coli RNA polymerase core enzyme studied by surface plasmon resonance

Abstract

The interaction between the core form of bacterial RNA polymerases and c c factors is essential for specific promoter recognition, and for coordinating the expression of different sets of genes in response to varying cellular needs. The interaction between Escherichia coli core RNA polymerase and c c 70 has been investigated by surface plasmon resonance. The His-tagged form of c c 70 factor was immobilised on a Ni 2+^N TA chip for monitoring its interaction with core polymerase. The binding constant for the interaction was found to be 1.9U U10^7 M, and the dissociation rate constant for release of c c from core, in the absence of DNA or transcription, was 4U U10^3 s^1, corresponding to a half-life of about 200 s. ß

Surface Plasmon Resonance – More Than a Screening Technology: Insights in the Binding Mode of σ 70 :Core Rnap Inhibitors

“…The equilibrium dissociation constant was determined to be 155 nM (Figure 1, for kinetics see Supplementary Figure 1). This K D is consistent with the dissociation constant of 190 nM determined for the σ 70 :core interaction by Ferguson et al in a different setup [34]. They immobilized the σ 70 factor through the His 6 -tag and injected the core enzyme.…”

Interaction of the CopZ Copper Chaperone with the CopA Copper ATPase of Enterococcus hirae Assessed by Surface Plasmon Resonance

“…For the interaction between the E. coli Hsp70 family member, DnaK, and its partner DnaJ, a ka of 2.3-3 ϫ 10 4 M Ϫ1 s Ϫ1 and a k d of 1.6 -3 ϫ 10 Ϫ3 s Ϫ1 was determined (28). Also using SPR analysis, the E. coli -sigma factor was found to interact with RNA polymerase with an association rate of 2.1 ϫ 10 4 M Ϫ1 s Ϫ1 and a dissociation rate of 4 ϫ 10 Ϫ3 s Ϫ1 (29). It is apparent that these widely different systems exhibit similar rate constants.…”

Studying the Salt Dependence of the Binding of σ70 and σ32 to Core RNA Polymerase Using Luminescence Resonance Energy Transfer

“…There are many ways in which protein-protein interactions can be measured. Non-homogeneous techniques such as surface plasmon resonance [1], pull-down assays (ex. co-immunoprecipitation), enzyme-linked immunosorbent assay (ELISA) [2], size exclusion chromatography [3], and glycerol gradient ultracentrifugation [4] all utilize separation steps that can result in the inaccurate measurements of protein-protein interactions when the transient interactions or weak interactions have half-lives shorter than the time needed for separation to occur.…”