Institute of Physical and Theoretical Chemistry and Center of Biomolecular Magnetic Resonance, Goethe University Frankfurt, 60438 Frankfurt am Main, Germany.
Department of Biology, Technical University of Darmstadt, 64287 Darmstadt, Germany.
RNA. 2019 Jan;25(1):158-167. doi: 10.1261/rna.068684.118. Epub 2018 Oct 18.
The tetracycline-binding RNA aptamer (TC-aptamer) is a synthetic riboswitch that binds the antibiotic tetracycline (TC) with exceptionally high affinity. Although a crystal structure exists of the TC-bound state, little is known about the conformational dynamics and changes upon ligand binding. In this study, pulsed electron paramagnetic resonance techniques for measuring distances (PELDOR) in combination with rigid nitroxide spin labels (Çm spin label) were used to investigate the conformational flexibility of the TC-aptamer in the presence and absence of TC at different Mg concentrations. TC was found to be the essential factor for stabilizing the tertiary structure at intermediate Mg concentrations. At higher Mg concentrations, Mg alone is sufficient to stabilize the tertiary structure. In addition, the orientation of the two spin-labeled RNA helices with respect to each other was analyzed with orientation-selective PELDOR and compared to the crystal structure. These results demonstrate for the first time the unique value of the Çm spin label in combination with PELDOR to provide information about conformational flexibilities and orientations of secondary structure elements of biologically relevant RNAs.
四环素结合 RNA 适体（TC-aptamer）是一种合成的核酶，能够以极高的亲和力结合抗生素四环素（TC）。尽管已经存在 TC 结合态的晶体结构，但对于配体结合时的构象动力学和变化知之甚少。在这项研究中，使用脉冲电子顺磁共振技术（PELDOR）结合刚性氮氧自由基自旋标记（Çm 自旋标记）来研究 TC-aptamer 在不同 Mg 浓度下存在和不存在 TC 时的构象灵活性。研究发现，TC 是在中间 Mg 浓度下稳定三级结构的必要因素。在更高的 Mg 浓度下，仅 Mg 就足以稳定三级结构。此外，还通过对选择性取向 PELDOR 进行分析并与晶体结构进行比较，研究了两个自旋标记 RNA 螺旋之间的相对取向。这些结果首次证明了Çm 自旋标记与 PELDOR 结合在提供有关生物相关 RNA 的二级结构元件的构象灵活性和取向方面的独特价值。
