Probing DNA Binding, DNA Opening, and Assembly of a Downstream Clamp/Jaw in <i>Escherichia coli</i> RNA Polymerase−λP<sub>R</sub> Promoter Complexes Using Salt and the Physiological Anion Glutamate Wayne S. Kontur Michael W. Capp Theodore J. Gries Ruth M. Saecker M. Thomas Record 10.1021/bi100092a.s001 https://acs.figshare.com/articles/journal_contribution/Probing_DNA_Binding_DNA_Opening_and_Assembly_of_a_Downstream_Clamp_Jaw_in_i_Escherichia_coli_i_RNA_Polymerase_P_sub_R_sub_Promoter_Complexes_Using_Salt_and_the_Physiological_Anion_Glutamate/2767126 Transcription by all RNA polymerases (RNAPs) requires a series of large-scale conformational changes to form the transcriptionally competent open complex RP<sub>o</sub>. At the λP<sub>R</sub> promoter, <i>Escherichia coli</i> σ<sup>70</sup> RNAP first forms a wrapped, closed 100 bp complex I<sub>1</sub>. The subsequent step opens the entire DNA bubble, creating the relatively unstable (open) complex I<sub>2</sub>. Additional conformational changes convert I<sub>2</sub> to the stable RP<sub>o</sub>. Here we probe these events by dissecting the effects of Na<sup>+</sup> salts of Glu<sup>−</sup>, F<sup>−</sup>, and Cl<sup>−</sup> on each step in this critical process. Rapid mixing and nitrocellulose filter binding reveal that the binding constant for I<sub>1</sub> at 25 °C is ∼30-fold larger in Glu<sup>−</sup> than in Cl<sup>−</sup> at the same Na<sup>+</sup> concentration, with the same log−log salt concentration dependence for both anions. In contrast, both the rate constant and equilibrium constant for DNA opening (I<sub>1</sub> to I<sub>2</sub>) are only weakly dependent on salt concentration, and the opening rate constant is insensitive to replacement of Cl<sup>−</sup> with Glu<sup>−</sup>. These very small effects of salt concentration on a process (DNA opening) that is strongly dependent on salt concentration in solution may indicate that the backbones of both DNA strands interact with polymerase throughout the process and/or that compensation is present between ion uptake and release. Replacement of Cl<sup>−</sup> with Glu<sup>−</sup> or F<sup>−</sup> at 25 °C greatly increases the lifetime of RP<sub>o</sub> and greatly reduces its salt concentration dependence. By analogy to Hofmeister salt effects on protein folding, we propose that the excluded anions Glu<sup>−</sup> and F<sup>−</sup> drive the folding and assembly of the RNAP clamp/jaw domains in the conversion of I<sub>2</sub> to RP<sub>o</sub>, while Cl<sup>−</sup> does not. Because the Hofmeister effect of Glu<sup>−</sup> or F<sup>−</sup> largely compensates for the destabilizing Coulombic effect of any salt on the binding of this assembly to downstream promoter DNA, RP<sub>o</sub> remains long-lived even at 0.5 M Na<sup>+</sup> in Glu<sup>−</sup> or F<sup>−</sup> salts. The observation that Eσ<sup>70</sup> RP<sub>o</sub> complexes are exceedingly long-lived at moderate to high Glu<sup>−</sup> concentrations argues that Eσ<sup>70</sup> RNAP does not dissociate from strong promoters in vivo when the cytoplasmic glutamate concentration increases during osmotic stress. 2010-05-25 00:00:00 DNA opening Physiological Anion GlutamateTranscription λ PR promoter nitrocellulose filter binding E σ70 RNAP Hofmeister salt effects 0.5 M Na salt concentration Cl salt concentration dependence cytoplasmic glutamate concentration increases Escherichia coli σ70 RNAP E σ70 RPo complexes