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Thermodynamics of stabilization of RNA pseudoknots by cobalt(III) hexaammine
Author(s) -
Nixon Paul L.,
Theimer Carla A.,
Giedroc David P.
Publication year - 1999
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/(sici)1097-0282(19991005)50:4<443::aid-bip9>3.0.co;2-c
Subject(s) - chemistry , crystallography , divalent , rna , delocalized electron , affinities , metal , cobalt , stereochemistry , inorganic chemistry , biochemistry , gene , organic chemistry
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 ions in solution. In this report, the apparent affinities of exchange inert trivalent Co(NH 3 ) 3+ 6have been determined, relative to divalent Mg 2+ , for the folded, partially folded ( K f ), and fully unfolded ( K u ) conformations of these molecules. A general nonspecific, delocalized ion binding model was developed and applied to the analysis of the metal ion concentration dependence of individual two‐state unfolding transitions. Trivalent Co(NH 3 ) 3+ 6was found to associate with the fully folded and partially unfolded pseudoknotted forms of these RNAs with a K f of 5–8 × 10 4 M −1 in a background of 0.10 M K + , or 3‐ to 5‐fold larger than the K f obtained for two model RNA hairpins and hairpin unfolding intermediates, and ≈ 40–50‐fold larger than K f for Mg 2+ . The magnitude of K f was found to be strongly dependent on the monovalent salt concentration in a manner qualitatively consistent with polyelectrolyte theory, with K f reaching 1.2 × 10 5 M −1 in 50 m M K + . Two RNA hairpins were found to have affinities for Co(NH 3 ) 3+ 6and Ru(NH 3 ) 3+ 6of 1–2 ×10 4 M −1 , or ≈ 15‐fold larger than the K f of ∼ 1000 M −1 observed for Mg 2+ . Additionally, the K u of 4,800 M −1 for the trivalent ligands is ≈ 8‐fold larger than the K u of 600 M −1 observed for Mg 2+ . These findings suggest that the T2 and T4 gene 32 mRNA pseudoknots possess a site(s) for Mg 2+ and Co(NH 3 ) 3+ 6binding of significantly higher affinity than a “duplexlike” delocalized ion binding site that is strongly linked to the thermodynamic stability of these molecules. Imino proton perturbation nmr spectroscopy suggests that this site(s) lies near the base of the pseudoknot stem S2, near a patch of high negative electrostatic potential associated with the region where the single loop L1 adenosine crosses the major groove of stem S2. © 1999 John Wiley & Sons, Inc. Biopoly 50: 443–458, 1999