Quantitative Analysis of the Isolated GAAA Tetraloop/Receptor Interaction in Solution: A Site-Directed Spin Labeling Study

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• 作者：Peter Z. Qin ; Samuel E. Butcher ; Juli Feigon ; and Wayne L. Hubbell
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：June 12, 2001
• 年：2001
• 卷：40
• 期：23
• 页码：6929 - 6936
• 全文大小：88K
• 年卷期：v.40,no.23(June 12, 2001)
• ISSN：1520-4995

文摘

The GNRA (N: any nucleotide; R: purine) tetraloop/receptor interaction is believed to beone of the most frequently occurring tertiary interaction motifs in RNAs, but an isolated tetraloop/receptorcomplex has not been identified in solution. In the present work, site-directed spin labeling is applied todetect tetraloop/receptor complex formation and estimate the free energy of interaction. For this purpose,the GAAA tetraloop/receptor interaction was chosen as a model system. A method was developed toplace nitroxide labels at specific backbone locations in an RNA hairpin containing the GAAA tetraloop.Formation of the tetraloop/receptor complex was monitored through changes in the rotational correlationtime of the tetraloop and the attached nitroxide. Results show that a hairpin containing the GAAA tetraloopforms a complex with an RNA containing the 11-nucleotide GAAA tetraloop receptor motif with anapparent K_d that is strongly dependent on Mg²⁺. At 125 mM MgCl₂, K_d = 0.40 ± 0.05 mM. Thecorresponding standard free energy of complex formation is -4.6 kcal/mol, representing the energeticsof the tetraloop/receptor interaction in the absence of other tertiary constraints. The experimental strategypresented here should have broad utility in quantifying weak interactions that would otherwise beundetectable, for both nucleic acids and nucleic acid-protein complexes.