Energetics and cooperativity of tertiary hydrogen bonds in RNA structure

Tertiary interactions that allow RNA to fold into intricate three-dimension al structures are being identified, but little is known about the thermodyn amics of individual interactions. Here we quantify the tertiary structure c ontributions of individual hydrogen bonds in a "ribose zipper" motif of the recently crystallized Tetrahymena group I intron P4-P6 domain. The 2'-hydr oxyls of P4-P6 nucleotides C109/A184 and A183/G110 participate in forming t he "teeth" of the zipper. These four nucleotides were substituted in all co mbinations with their 2'-deoxy and (separately) 2'-methoxy analogues, and t hermodynamic effects on the tertiary folding Delta G degrees' were assayed by the Mg2+ dependence of electrophoretic mobility in nondenaturing gels. T he 2'-deoxy series showed a consistent trend with an average contribution t o the tertiary folding Delta G degrees' of -0.4 to -0.5 kcal/mol per hydrog en bond. Contributions were approximately additive, reflecting no cooperati vity among the hydrogen bonds. Each "tooth" of the ribose zipper (comprisin g two hydrogen bonds) thus contributes about -1.0 kcal/mol to the tertiary folding Delta G degrees'. Single 2'-methoxy substitutions destabilized fold ing by similar to 1 kcal/mol, but the trend reversed with multiple 2'-metho xy substitutions; the folding Delta G degrees' 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 en tropically favorable hydrophobic interactions balance enthalpically unfavor able hydrogen bond deletions and steric clashes for multiple 2'-methoxy sub stitutions. Because many of the 2'-deoxy derivatives no longer have the cha racteristic hydrogen-bond patterns of the ribose zipper motif but simply ha ve individual long-range ribose-base or ribose-ribose hydrogen bonds, we sp eculate that the energetic value of -0.4 to -0.5 kcal/mol per tertiary hydr ogen bond may be more generally applicable to RNA folding.