ROLES OF MG2+ IN THE MECHANISM OF FORMATION AND DISSOCIATION OF OPEN COMPLEXES BETWEEN ESCHERICHIA-COLI RNA-POLYMERASE AND THE LAMBDA-PR PROMOTER - KINETIC EVIDENCE FOR A 2ND OPEN COMPLEX REQUIRING MG2+

Comparative studies of the effects of Mg2+ vs Na+ and of acetate (OAc-) vs Cl- on the kinetics of formation and dissociation of E. coli RNA polymerase (E-sigma-70)-lambda-P(R) promoter open complexes have been used to probe the mechanism of this interaction. Composite second-order association rate constants k(a) and first-order dissociation rate constants k(d), and their power dependences on salt concentration Sk(a) (Sk(a) = d log k(a)/d log [salt]) and Sk(d) (Sk(d) = d log k(d)/d log [salt]), were determined in MgCl2 and NaOAc to compare with the results of Roe and Record (1985) in NaCl. Replacement of NaCl by MgCl2 reduces the magnitude of Sk(a) 2-fold (Sk(a) = -11.9 +/- 1.1 in NaCl; Sk(a) = -5.2 +/- 0.3 in MgCl2) and (by extrapolation) drastically reduces the magnitude of k(a) at any specified salt concentration (e.g., approximately 10(6)-fold at 0.2 M). Replacement of NaCl by NaOAc does not significantly affect Sk(a) (Sk(a) = -12.0 +/- 0.7 in NaOAc) and (by extrapolation) increases k(a) by approximately 80-fold at any fixed [Na+]. In the absence of Mg2+, replacement of NaCl by NaOAc is found to increase the half-life of the open complex by approximately 560-fold at fixed [Na+] without affecting Sk(d) [Sk(d) = 7.6 +/- 0.1 in NaOAc; in NaCl, Sk(d) = 7.7 +/- 0.2 (Roe & Record, 1985)]. Replacement of NaCl by MgCl2 drastically reduces both Sk(d) and the half-life of the open complex at any salt concentration below approximately 0.2 M. Strikingly, Sk(d) = 0.4 +/- 0.1 in MgCl2, indicating that the net uptake of Mg2+ ions in the kinetically significant steps in dissociation of the open complex is much smaller than that expected by analogy with the uptake of approximately 8 Na+ ions in the corresponding steps in NaCl. In NaCl/MgCl2 mixtures, at a constant [NaCl] in the range 0.1-0.2 M, initial addition of MgCl2 (0.5 mM less-than-or-equal-to [MgCl2] less-than-or-equal-to 1 mM) increases the half-life of the open complex; further addition Of MgCl2 causes the half-life to decrease, though the effect Of [MgCl2] on k(d) is always less than that predicted by a simple competitive model. The observed effects Of MgCl2 on Sk(d) and k(d) differ profoundly from those expected from the behavior of k(d) and Sk(d) in NaCl and NaOAc and indicate that the role of Mg2+ in dissociation is not merely that of a nonspecific divalent competitor with RNAP for interactions with DNA phosphates and of a DNA helix-stabilizer, both of which should cause k(d) to increase monotonically with increasing [Mg2+]. These observations provide evidence for differences in the nature of the open complex at the lambda-P(R) promoter in the presence and absence of Mg2+ and lead us to propose the existence of at least two mechanistically significant forms of the open complex in the presence of Mg2+ (RP(o1), RP(o2)). We deduce that the conversion of RP(o1) to RP(o2) requires uptake of approximately 3 Mg2+ ions, presumably at specific sites on RNA polymerase, and does not occur in the absence of Mg2+ (i.e., in NaCl or NaOAc). We therefore propose a minimal four-step mechanism in which the specific uptake of Mg2+ upon conversion of RP(o1), to RP(o2) contrasts with nonspecific cation accumulation (either Na+ or Mg2+, as shown) in the dissociation of the initial closed complex (RP(c1) to free promoter DNA (P) and RNA polymerase (R), and in the closing (DNA helix formation) of the initial open complex (RP(o1)) to form RP(c2), the intermediate closed complex which differs from RP(c1) as the result of a large conformational change in RNA polymerase. [GRAPHICS] According to this mechanism, the open complex RP(o1) (formed in the absence or presence of Mg2+) is an obligate intermediate in formation of RP(o2), the open complex which initiates transcription upon binding nucleotides.