EFFECTS OF MG2+, K+, AND H+ ON AN EQUILIBRIUM BETWEEN ALTERNATIVE CONFORMATIONS OF AN RNA PSEUDOKNOT

A complex pseudoknot structure surrounds the first ribosome initiation site in the Eschericlia coli alpha mRNA and mediates its regulation b y ribosomal protein S4. A 112 nt RNA fragment containing this pseudokn ot exists in two conformations that are resolvable by gel electrophore sis below room temperature. Between 30 degrees C and 45 degrees C the conformers reach thermodynamic equilibrium on a time scale ranging fro m one hour to one minute, and the interconversion between conformers i s Linked to H+, K+ and Mg2+ concentrations. Mg2+ favors formation of t he ''fast'' electrophoretic form: a single Mg2+ is bound in the rate-L imiting step, followed by cooperative binding of similar to 1.7 additi onal ions. Binding of the latter ions provides most of the favorable f ree energy for the reaction. However, the ''slow'' form binds about th e same number of Mg ions, albeit more weakly, so that saturating Mg2concentrations drive the equilibrium to only similar to 70% fast form. A single H+ is taken up in the switch to the ''slow'' conformer, whic h has apparent pK approximate to 5.9; low pH also stabilizes part of t he pseudoknot structure melting at similar to 62 degrees C. Mg2+ and H + appear to direct alpha mRNA folding by relatively small (10 to 100-f old) differences in their affinities for alternative conformers. K+ ha s very Little effect on the conformational equilibrium, but at high co ncentrations accelerates interconversion between the conformers. The a lpha mRNA conformational switch is similar in its slow kinetics, large activation energy, and Mg2+ dependence of the equilibrium constant to slow steps in the folding of tRNA, group I introns, and RNase P RNA t ertiary structures, though it differs from these in the association of a single Mg2+ with the rate-limiting step. (C) 1997 Academic Press Li mited.