A Semiconserved Residue Inhibits Complex Formation by Stabilizing Interactions in the Free State of a Theophylline-Binding RNA
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- 作者：Grant R. Zimmermann ; Thomas P. Shields ; Robert D. Jenison ; Catherine L. Wick ; and Arthur Pardi
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：June 23, 1998
- 年：1998
- 卷：37
- 期：25
- 页码：9186 - 9192
- 全文大小：159K
- 年卷期：v.37,no.25(June 23, 1998)
- ISSN：1520-4995
文摘
The theophylline-binding RNA aptamer contains a 15 nucleotidemotif that is required forhigh-affinity ligand binding. One residue within this RNA motif isonly semiconserved and can be an Aor C. This residue, C27, was disordered in the previouslydetermined three-dimensional structure of thecomplex, suggesting that it is dynamic in solution.13C Relaxation measurements are reportedhere,demonstrating that C27 is highly dynamic in the otherwise well-orderedRNA-theophylline complex. Asynthetic complex with an abasic residue at position 27 was found toexhibit wild-type binding affinity(Kd ~ 0.2 
M), indicating that the base ofresidue 27 is not directly involved with theophyllinebinding.Surprisingly, the U27 and G27 RNAs were found to bind theophyllinewith low affinity (Kd values > 4M). NMR spectroscopy on the U27 RNA revealed the presence of anA7-U27 base pair in the freeRNA that prevents formation of a critical base-platform structuralmotif and therefore blocks theophyllinebinding. Similarly, a protonated A7H+-C27 base pairforms in the absence of theophylline at low pH,which explains the unusual pH dependence of theophylline binding of theC27 RNA aptamer. Thus theweak binding for various nucleotides at position 27 arises not fromunfavorable interactions in the RNA-theophylline complex but instead from stable interactions in the freestate of the RNA that inhibittheophylline binding.
M), indicating that the base ofresidue 27 is not directly involved with theophyllinebinding.Surprisingly, the U27 and G27 RNAs were found to bind theophyllinewith low affinity (Kd values > 4M). NMR spectroscopy on the U27 RNA revealed the presence of anA7-U27 base pair in the freeRNA that prevents formation of a critical base-platform structuralmotif and therefore blocks theophyllinebinding. Similarly, a protonated A7H+-C27 base pairforms in the absence of theophylline at low pH,which explains the unusual pH dependence of theophylline binding of theC27 RNA aptamer. Thus theweak binding for various nucleotides at position 27 arises not fromunfavorable interactions in the RNA-theophylline complex but instead from stable interactions in the freestate of the RNA that inhibittheophylline binding.