Equivalence of Mg and Na ions in salt dependence of the equilibrium binding and dissociation rate constants of RNA polymerase open complex

Tomasz Łoziński , Krystyna Bolewska , Kazimierz L. Wierzchowski

Conflicting experimental data on the influence of Mg ions on the salt dependence of formation/dissociation of open transcription complex (RPo) of RNA polymerase led us to carry systematic measurements of the dissociation rate constant () and thermodynamic stability of complexes at λP and Pa promoters in a broad range of [NaCl] and [MgCl] at 25, 31 and 37°C, using fluorescence detected abortive transcription assay. Values of determined in MgCl in the presence of heparin, as a commonly used anionic competitor, were shown to depend on heparin concentration whereas in NaCl this effect was not observed. Kinetics of dissociation was therefore determined in the course of salt-induced down-shift of the binding equilibrium. Salt derivatives of 's (n) appeared to be similar in NaCl (∼8.5) and MgCl (∼10) for both complexes. Isotherms of fractional occupancy of promoters by RNAP as a function of ln [salt] were shown to conform to a sigmoid Boltzman function parameterized to include binding constant of RPo and a net change (n) in the number of electrolyte ions associated with complex components upon its formation/dissociation. The fitted values of n appeared also similar in NaCl and in MgCl: ∼18 for RPo/λP and ∼20 for RPo/Pa, respectively. Overall unfavorable vant'Hoff enthalpy (ΔH) of RPo proved to be much higher in MgCl than in NaCl by ca. 20 kcal/mol for both complexes, rendering them profoundly less stable in the former salt. In both salts, ΔH was higher by ∼30 kcal/mol for RPo/Pa relative to RPo/λP. Similarity of n and n values for the two salts indicates thermodynamic equivalence of Mg and Na in [salt]-controlled binding equilibrium of RPo. This finding remains in disagreement with earlier data and suggests that salt effects on open complex stability should be sought in global compensating changes in distribution of all ionic species around the interacting complex components.

Mots clés: RNA polymerase; open transcription complex; λP promoter; salt dependence of open complex stability; effect of Mg on open complex stability

Tomasz Łoziński, Krystyna Bolewska, Kazimierz L. Wierzchowski. Equivalence of Mg and Na ions in salt dependence of the equilibrium binding and dissociation rate constants of RNA polymerase open complex. Biophysical Chemistry, 2009, 142 (1-3), pp.65. ⟨10.1016/j.bpc.2009.03.004⟩. ⟨hal-00528544⟩