Stabilization of RNA tertiary structure by monovalent cations

The effects of monovalent cations (Li+, Na+, K+, Rb+, Cs+, and NH4(+)) on the thermal stability of RNA tertiary structure were investigated by UV melting. We show that with the RNA used here (nucleotides 1051-1108 of Escherichia coli 23 S rRNA with four base substitutions), monovalent cations and Mg2+ compete in stabilizing the RNA tertiary structure, and that the competition takes place between two boundaries: one where Mg2+ concentration is zero and the other where it is maximally stabilizing ("saturating"). The pattern of competition is the same for all monovalent cations and depends on the cation's ability to displace Mg2+ from the RNA, its ability to stabilize tertiary structure in the absence of Mg2+, and its ability to stabilize tertiary structure at saturating Mg2+ concentrations. The stabilizing ability of a monovalent cation depends on its unhydrated ionic radius, and at a low monovalent cation concentration and saturating Mg2+, there is a (calculated) net release of a single monovalent cation/RNA molecule when tertiary structure is denatured. The implications are that under these conditions there is at least one binding site for monovalent cations on the RNA, the site is specifically associated with formation of stable tertiary structure, K+ is the most effective of the tested cations, and Mg2+ appears ineffective at this site. At high ionic strength, and in the absence of Mg2+, stabilization of tertiary structure is still monovalent-cation specific and ionic-radius dependent, but a larger number of cations (similar to eight) are released upon RNA tertiary structure denaturation, and NH4+ appears to be the most effective cation in stabilizing tertiary structure under these conditions. In the majority of the experiments, methanol was added as a cosolvent to the buffer. Its use allowed the examination of the behavior of monovalent ions under conditions where their effects would otherwise have been too weak to be observed. Methanol stabilizes tertiary but not secondary structure of the RNA. There was no evidence that it either causes qualitative changes in cation-binding properties of the RNA or a change in the pattern of monovalent cation/Mg2+ competition. (C) 2000 Academic Press.