Thermodynamics of stabilization of RNA pseudoknots by cobalt(III) hexaammine

Equilibrium unfolding (folding) studies reveal that the autoregulatory RNA pseudoknots derived from the bacteriophage T2 and T4 gene 32 mRNAs exhibit significant stabilization by increasing concentrations of divalent metal io ns in solution. In this report, the apparent affinities of exchange inert t rivalent Co(NH3)(6)(3+) have been determined, relative to divalent Mg2+, fo r the folded, partially folded (K-f), and fully unfolded (K-u) conformation s of these molecules. A general nonspecific, delocalized ion binding model was developed and applied to the analysis of the metal ion concentration de pendence of individual two-slate unfolding transitions. Trivalent Co(NH3)(6 )(3+) was found to associate with the Stilly folded and partially unfolded pseudoknotted forms of these RNAs with a K-f of 5-8 x 10(4) M-1 in a backgr ound of 0.10 M K+, or 3- to 5-fold larger than the K-f obtained for two mod el RNA hairpins and hairpin unfolding intermediates, and approximate to 40- 50-fold larger than K-f for Mg2+. The magnitude of K-f was found to be stro ngly dependent on the monovalent salt concentration in a manner qualitative ly consistent with polyelectrolyte theory, with K-f reaching 1.2 x 10(5) M- 1 in 50 mM K+. TWO RNA hairpins were found to have affinities for Co(NH3)(6 )(3+) and Ru(NH3)(6)(3+) of 1-2 x 10(4) M-1 or approximate to 15-fold large r than the K-f of similar to 1000 M-1 observed for Mg2+. Additionally the K -u of 4,800 M-1 for the trivalent ligands is approximate to 8-fold larger t han the K-u of 600 M-1 observed for Mg2+. These findings suggest that the T 2 and T4 gene 32 mRNA pseudoknots possess a site(s)for Mg2+ and Co(NH3)(6)( 3+) binding of significantly higher affinity than a "duplex-like" delocaliz ed ion binding site that is strongly linked to the thermodynamic stability of these molecules. Imino proton perturbation nmr spectroscopy suggests tha t this site(s) lies near the base of the pseudoknot stem S2, near a patch o f high negative electrostatic potential associated with the region where th e single loop L1 adenosine crosses the major groove of stem S2. (C) 1999 Jo hn Wiley & Sons, Inc.