Quantitative analysis of the isolated GAAA tetraloop/receptor interaction in solution: A site-directed spin labeling study
Quantitative analysis of the isolated GAAA tetraloop/receptor interaction in solution: A site-directed spin labeling study
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DOI：
10.1021/bi010294g
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发表时间：
2001-06-12
期刊：
影响因子：
2.9
通讯作者：
Hubbell, WL
中科院分区：
文献类型：
--
作者：
Qin, PZ;Butcher, SE;Hubbell, WL
GNRA（N：任何核苷酸; R：嘌呤）四环/受体相互作用被认为是RNA中最常见的三级相互作用基序之一，但尚未在溶液中鉴定出分离的四环/受体复合物。在目前的工作中，定点自旋标记应用于检测四环/受体复合物的形成和估计相互作用的自由能。为此，选择GAAA四环/受体相互作用作为模型系统。开发了一种将氮氧自由基标记物放置在含有GAAA四环的RNA发夹中的特定骨架位置的方法。四环/受体复合物的形成通过四环和所附氮氧自由基的旋转相关时间的变化进行监测。结果表明，含有GAAA四环的发夹与含有II-核苷酸GAAA四环受体基序的RNA形成复合物，其具有强烈依赖于Mg 2+的表观Kd。在125 mM MgCl 2下，Kd = 0.40 +/-0.05 mM。复合物形成的相应标准自由能为-4.6 kcal/mol，代表在不存在其它三级约束的情况下四环/受体相互作用的能量学。本文提出的实验策略在定量核酸和核酸-蛋白质复合物的弱相互作用方面应该具有广泛的实用性，否则这些弱相互作用是不可检测的。
The GNRA (N: any nucleotide; R: purine) tetraloop/receptor interaction is believed to be one of the most frequently occurring tertiary interaction motifs in RNAs, but an isolated tetraloop/receptor complex has not been identified in solution. In the present work, site-directed spin labeling is applied to detect 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 to place 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 correlation time of the tetraloop and the attached nitroxide. Results show that a hairpin containing the GAAA tetraloop forms a complex with an RNA containing the Il-nucleotide GAAA tetraloop receptor motif with an apparent Kd that is strongly dependent on Mg2+. At 125 mM MgCl2, K-d = 0.40 +/- 0.05 mM. The corresponding standard free energy of complex formation is -4.6 kcal/mol, representing the energetics of the tetraloop/receptor interaction in the absence of other tertiary constraints. The experimental strategy presented here should have broad utility in quantifying weak interactions that would otherwise be undetectable, for both nucleic acids and nucleic acid -protein complexes.