Thermodynamics of ion-induced RNA folding in the hammerhead ribozyme: an isothermal titration calorimetric study.
The hammerhead ribozyme undergoes a well-defined two-stage conformational folding process, induced by the binding of magnesium ions. In this study, we have used isothermal titration calorimetry to analyze the thermodynamics of magnesium binding and magnesium ion-induced folding of the ribozyme. Binding to the natural sequence ribozyme is strongly exothermic and can be analyzed in terms of sequential interaction at two sites with association constants K(A) = 480 and 2840 M(-1). Sequence variants of the hammerhead RNA give very different isothermal titration curves. An A14G variant that cannot undergo ion-induced folding exhibits endothermic binding. By contrast, a deoxyribose G5 variant that can undergo only the first of the two folding transitions gives a complex titration curve. However, despite these differences the ITC data for all three species can be analyzed in terms of the sequential binding of magnesium ions at two sites. While the binding affinities are all in the region of 10(3) M(-1), corresponding to free energies of Delta G degrees = -3.5 to -4 kcal mol(-1), the enthalpic and entropic contributions show much greater variation. The ITC experiments are in good agreement with earlier conformational studies of the folding of the ion-induced folding of the hammerhead ribozyme.
Hammann
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C
CRC Nucleic Acid Structure Research Group, Department of Biochemistry, The University of Dundee, Dundee DD1 4HN, UK.
Cooper
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A
Lilley
D M
DM
D000225:Adenine / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002151:Calorimetry; D002413:Cations, Divalent / Q000737:chemistry; D003849:Deoxyguanosine / Q000737:chemistry; D014644:Genetic Variation; D006147:Guanine / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry*; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics; D016053:RNA, Protozoan / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics; D017186:Titrimetry
D002413:Cations, Divalent; D016337:RNA, Catalytic; D016053:RNA, Protozoan; D006147:Guanine; D003849:Deoxyguanosine; D008274:Magnesium; D000225:Adenine