Affinities and selectivities of divalent cation binding sites within an RNA tertiary structure

Abstract

A 58 nucleotide fragment of Escherichia coli large subunit ribosomal RNA, nucleotides 1051 to 1108, adopts a specific tertiary structure normally requiring both monovalent (NH4+ or K+) and divalent (Mg2+) ions to fold; this ion-dependent structure is a prerequisite for recognition by ribosomal protein L11. Melting experiments have been used to show that a sequence variant of this fragment, GACG RNA, is able to adopt a stable tertiary structure in the presence of 1.6 M NH4Cl and absence of divalent ions. The similarity of this high-salt structure to the tertiary structure formed under more typical salt conditions (0.1 M NH4Cl and several mM MgCl2) was shown by its following properties: (i) an unusual ratio of hyperchromicity at 260 nm and 280 nm upon unfolding, (ii) selectivity for NH4+ over K+ or Na+, (iii) stabilization by L11 protein, and (iv) further stabilization by added Mg2+. Delocalized electrostatic interactions of divalent ions with nucleic acids should be very weak in the presence of >1 M monovalent salt; thus stabilization of the tertiary structure by low (<1 mM) Mg2+ concentrations in these high-salt conditions suggests that Mg2+ binds at specific site(s).GACG RNA tertiary structure unfolding in 1.6 M NH4Cl (Tm ≈ 39°C) is distinct from melting of the secondary structure (centered at ∼72°C), and it has been possible to calculate the free energy of tertiary structure stabilization upon addition of various divalent cations. From these binding free energies, ion-RNA binding isotherms for Mn2+, Mg2+, Ca2+, Sr2+ and Ba2+have been obtained. All of these ions bind at two sites: one site favors Mg2+ and Ba2+ and discriminates against Ca2+, while the other site favors binding of smaller ions over larger ones (Mg2+ > Ca2+ > Sr2+ > Ba2+). Weak cooperative or anticooperative interactions between the sites, also dependent on ion radius, may also be taking place.