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 Escherichia coli alpha 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 degrees C and 45 degrees 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 Mg2+ concentrations. Mg2+ favors formation of the "fast" electrophoretic form: a single Mg2+ is bound in the rate-limiting step, followed by cooperative binding of approximately 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 Mg2+ concentrations drive the equilibrium to only approximatley 70% fast form. A single H+ is taken up in the switch to the "slow" conformer, which has apparent pK approximately 5.9; low pH also stabilizes part of the pseudoknot structure melting at approximately 62 degrees C. Mg2+ and H+ appear to direct alpha 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 alpha 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 tertiary structures, though it differs from these in the association of a single Mg2+ with the rate-limiting step.
Gluick
T C
TC
Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA.
Gerstner
R B
RB
Draper
D E
DE
D001483:Base Sequence; D002412:Cations / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008278:Magnesium Sulfate / Q000494:pharmacology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D011189:Potassium Chloride / Q000494:pharmacology; D012329:RNA, Bacterial / Q000737:chemistry*; D012333:RNA, Messenger / Q000737:chemistry*
D002412:Cations; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011189:Potassium Chloride; D008278:Magnesium Sulfate