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 DeltaG degrees ' 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 DeltaG degrees' 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 DeltaG degrees'. Single 2'-methoxy substitutions destabilized folding by approximately 1 kcal/mol, but the trend reversed with multiple 2'-methoxy substitutions; the folding DeltaG 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 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.
Silverman
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Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado at Boulder 80309-0215, USA. Scott.Silverman@colorado.edu
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D000818:Animals; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D019277:Entropy; D006860:Hydrogen Bonding; D006878:Hydroxides / Q000737:chemistry; D007700:Kinetics; D009154:Mutation; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry; D016053:RNA, Protozoan / Q000737:chemistry* / Q000235:genetics; D012266:Ribose / Q000737:chemistry; D013696:Temperature; D013768:Tetrahymena / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics
D006878:Hydroxides; D016337:RNA, Catalytic; D016053:RNA, Protozoan; D012266:Ribose; C031356:hydroxide ion