Department of Molecular Biosciences, Northwestern University, Evanston, IL, 60208, USA.
Department of Biochemistry and Molecular Biology and Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, 60637, USA.
Nat Commun. 2024 Aug 3;15(1):6592. doi: 10.1038/s41467-024-50885-x.
T-box riboswitches are noncoding RNA elements involved in genetic regulation of most Gram-positive bacteria. They regulate amino acid metabolism by assessing the aminoacylation status of tRNA, subsequently affecting the transcription or translation of downstream amino acid metabolism-related genes. Here we present single-molecule FRET studies of the Mycobacterium tuberculosis IleS T-box riboswitch, a paradigmatic translational T-box. Results support a two-step binding model, where the tRNA anticodon is recognized first, followed by interactions with the NCCA sequence. Furthermore, after anticodon recognition, tRNA can transiently dock into the discriminator domain even in the absence of the tRNA NCCA-discriminator interactions. Establishment of the NCCA-discriminator interactions significantly stabilizes the fully bound state. Collectively, the data suggest high conformational flexibility in translational T-box riboswitches; and supports a conformational selection model for NCCA recognition. These findings provide a kinetic framework to understand how specific RNA elements underpin the binding affinity and specificity required for gene regulation.
T 盒核糖体开关是涉及大多数革兰氏阳性菌遗传调控的非编码 RNA 元件。它们通过评估 tRNA 的氨酰化状态来调节氨基酸代谢，从而影响下游氨基酸代谢相关基因的转录或翻译。在这里，我们展示了结核分枝杆菌 IleS T 盒核糖体开关的单分子 FRET 研究，这是一个典型的翻译 T 盒。结果支持两步结合模型，其中首先识别 tRNA 的反密码子，然后与 NCCA 序列相互作用。此外，在反密码子识别之后，即使在没有 tRNA NCCA-鉴别子相互作用的情况下，tRNA 也可以短暂地停靠在鉴别子域中。NCCA-鉴别子相互作用的建立显著稳定了完全结合状态。总的来说，这些数据表明翻译 T 盒核糖体开关具有很高的构象灵活性；并支持 NCCA 识别的构象选择模型。这些发现为理解特定 RNA 元件如何为基因调控所需的结合亲和力和特异性提供了一个动力学框架。
