Effects of Mg 2+, K +, and H + on an equilibrium between alternative conformations of an RNA pseudoknot

Abstract

A complex pseudoknot structure surrounds the first ribosome initiation site in the Eschericlia coli α mRNA and mediates its regulation by ribosomal protein S4. A 112 nt RNA fragment containing this pseudoknot exists in two conformations that are resolvable by gel electrophoresis below room temperature. Between 30°C and 45°C the conformers reach thermodynamic equilibrium on a time scale ranging from one hour to one minute, and the interconversion between conformers is linked to H +, K + and Mg 2+ concentrations. Mg 2+ favors formation of the “fast” electrophoretic form: a single Mg 2+ is bound in the rate-limiting step, followed by cooperative binding of ∼1.7 additional ions. Binding of the latter ions provides most of the favorable free energy for the reaction. However, the “slow” form binds about the same number of Mg ions, albeit more weakly, so that saturating Mg 2+ concentrations drive the equilibrium to only ∼70% fast form. A single H + is taken up in the switch to the “slow” conformer, which has apparent pK≈5.9; low pH also stabilizes part of the pseudoknot structure melting at ∼62°C. Mg 2+ and H + appear to direct α mRNA folding by relatively small (10 to 100-fold) differences in their affinities for alternative conformers. K + has very little effect on the conformational equilibrium, but at high concentrations accelerates interconversion between the conformers. The α mRNA conformational switch is similar in its slow kinetics, large activation energy, and Mg 2+ dependence of the equilibrium constant to slow steps in the folding of tRNA, group I introns, and RNase P RNA tertiary structures, though it differs from these in the association of a single Mg 2+ with the rate-limiting step.