Energetics and Cooperativity of Tertiary Hydrogen Bonds in RNA Structure^†

Tertiary interactions that allow RNA to fold into intricate three-dimensional structures are being identified, but little is known about the thermodynamics of individual interactions. Here we quantify the tertiary structure contributions of individual hydrogen bonds in a “ribose zipper” motif of the recently crystallized Tetrahymena group I intron P4−P6 domain. The 2‘-hydroxyls of P4−P6 nucleotides C109/A184 and A183/G110 participate in forming the “teeth” of the zipper. These four nucleotides were substituted in all combinations with their 2‘-deoxy and (separately) 2‘-methoxy analogues, and thermodynamic effects on the tertiary folding ΔG°‘ were assayed by the Mg²⁺ dependence of electrophoretic mobility in nondenaturing gels. The 2‘-deoxy series showed a consistent trend with an average contribution to the tertiary folding ΔG°‘ of −0.4 to −0.5 kcal/mol per hydrogen bond. Contributions were approximately additive, reflecting no cooperativity among the hydrogen bonds. Each “tooth” of the ribose zipper (comprising two hydrogen bonds) thus contributes about −1.0 kcal/mol to the tertiary folding ΔG°‘. Single 2‘-methoxy substitutions destabilized folding by ∼1 kcal/mol, but the trend reversed with multiple 2‘-methoxy substitutions; the folding ΔG°‘ for the quadruple 2‘-methoxy derivative was approximately unchanged relative to wild-type. On the basis of these data and on temperature-gradient gel results, we conclude that entropically favorable hydrophobic interactions balance enthalpically unfavorable hydrogen bond deletions and steric clashes for multiple 2‘-methoxy substitutions. Because many of the 2‘-deoxy derivatives no longer have the characteristic hydrogen-bond patterns of the ribose zipper motif but simply have individual long-range ribose-base or ribose-ribose hydrogen bonds, we speculate that the energetic value of −0.4 to −0.5 kcal/mol per tertiary hydrogen bond may be more generally applicable to RNA folding.