Equivalence of Mg2+ and Na+ ions in salt dependence of the equilibrium binding and dissociation rate constants of Escherichia coli RNA polymerase open complex.
Conflicting experimental data on the influence of Mg(2+) ions on the salt dependence of formation/dissociation of open transcription complex (RPo) of Escherichia coli RNA polymerase led us to carry systematic measurements of the dissociation rate constant (k(d)) and thermodynamic stability of complexes at lambdaP(R) and Pa promoters in a broad range of [NaCl] and [MgCl(2)] at 25, 31 and 37 degrees C, using fluorescence detected abortive transcription assay. Values of k(d) determined in MgCl(2) 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 k(d)'s (n(d)) appeared to be similar in NaCl (approximately 8.5) and MgCl(2) (approximately 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(obs)) in the number of electrolyte ions associated with complex components upon its formation/dissociation. The fitted values of n(obs) appeared also similar in NaCl and in MgCl(2): approximately 18 for RPo/lambdaP(R) and approximately 20 for RPo/Pa, respectively. Overall unfavorable vant'Hoff enthalpy (DeltaH(obs)) of RPo proved to be much higher in MgCl(2) than in NaCl by ca. 20 kcal/mol for both complexes, rendering them profoundly less stable in the former salt. In both salts, DeltaH(obs) was higher by approximately 30 kcal/mol for RPo/Pa relative to RPo/lambdaP(R). Similarity of n(obs) and n(d) values for the two salts indicates thermodynamic equivalence of Mg(2+) 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.
Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland.
Tomasz
T
Loziński
Krystyna
K
Bolewska
Kazimierz L
KL
Wierzchowski
D001483:Base Sequence; D001665:Binding Sites / Q000187:drug effects; D002413:Cations, Divalent / Q000494:pharmacology; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology*; D008969:Molecular Sequence Data; D055550:Protein Stability; D012492:Salts / Q000494:pharmacology*; D012964:Sodium / Q000494:pharmacology*; D013816:Thermodynamics
D002413:Cations, Divalent; D012492:Salts; D008345:Manganese; D012964:Sodium; D012321:DNA-Directed RNA Polymerases