Single-molecule mechanical unfolding kinetics of unmodified Saccharomyces cerevisiae tRNAPhe: a hint to the tRNA chaperone-tRNA interaction mechanism
Single-molecule mechanical unfolding kinetics of unmodified Saccharomyces cerevisiae tRNAPhe: a hint to the tRNA chaperone-tRNA interaction mechanism
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DOI：
10.1101/2021.05.03.442431
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发表时间：
2021-05
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通讯作者：
Wenzhao Liu;L. Feng;Wenpeng Zhu;Zhenyu Zou;Ran Chen;Jie Zhou;W. Xie;Hu Chen;Zhensheng Zhong;Jie Ma
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作者：
Wenzhao Liu;L. Feng;Wenpeng Zhu;Zhenyu Zou;Ran Chen;Jie Zhou;W. Xie;Hu Chen;Zhensheng Zhong;Jie Ma
TRNA的生物学活性与其机械折叠特性密切相关。虽然以前的研究主要集中在tRNA的折叠和去折叠机制上，但其动力学在很大程度上是未知的。在这项研究中，结合光钳和分子动力学模拟，我们表征了单个未经修饰的酿酒酵母tRNAphe的机械折叠和去折叠过程。我们确定了在镁离子存在下tRNA机械折叠和去折叠的中间体和途径，发现D茎环和T茎环的折叠/去折叠动力学显著地受其上游和下游结构的影响，而反密码子茎环的折叠/去折叠动力学不受其上下游结构的显著影响。在镁离子存在下，tRNA的协同去折叠导致了折叠和去折叠途径之间的大滞后，这种滞后和去折叠协同效应可以通过降低镁离子浓度或突变形成‘肘状’结构的核苷酸来显著降低。此外，Steed分子动力学模拟和光钳实验结果都支持，肘部三级相互作用的形成增加了机械去折叠途径的能垒，包括中间体之间的能垒，并决定了整体去折叠协作性。我们的研究可能有助于揭示TRUB和TRMA的tRNA伴侣的详细机制。
The biological activity of tRNA is closely related to its mechanical folding properties. Although previous studies focused on the folding and unfolding mechanism of tRNA, its kinetics are largely unknown. In this study, combining optical tweezers and molecule dynamics simulations, we characterized the mechanical folding and unfolding processes of a single unmodified Saccharomyces cerevisiae tRNAphe. We identified the intermediates and pathways for tRNA mechanical folding and unfolding in the presence of Mg2+, discovering that the folding/unfolding kinetics of D stem-loop and T stem-loop but not the anti-codon stem-loop significantly affected by their upstream and downstream structures. The cooperative unfolding of the tRNA in the presence of Mg2+ lead to a large hysteresis between the folding and unfolding pathway, and such hysteresis and unfolding cooperativity are significantly reduced by lowering the Mg2+ concentration or mutating the nucleotides forming the ‘elbow’ structure. Moreover, both steered molecular dynamics simulation and optical tweezers experiment results support that, formation of tertiary interactions in the elbow region increases energy barriers of the mechanical unfolding pathway, including those in between intermediates, and determines the overall unfolding cooperativity. Our studies may shed light on the detailed tRNA chaperone mechanism of TruB and TrmA.