T. maritima and B. subtilis are bacteria that inhabit significantly different thermal environments, ∼80 vs. ∼40°C, yet employ similar lysine riboswitches to aid in the transcriptional regulation of the genes involved in the synthesis and transport of amino acids. Despite notable differences in G-C basepair frequency and primary sequence, the aptamer moieties of each riboswitch have striking similarities in tertiary structure, with several conserved motifs and long-range interactions. To explore genetic adaptation in extreme thermal environments, we compare the kinetic and thermodynamic behaviors in T. maritima and B. subtilis lysine riboswitches via single-molecule fluorescence resonance energy transfer analysis. Kinetic studies reveal that riboswitch folding rates increase with lysine concentration while the unfolding rates are independent of lysine. This indicates that both riboswitches bind lysine through an induced-fit (“bind-then-fold”) mechanism, with lysine binding necessarily preceding conformational changes. Temperature-dependent van’t Hoff studies reveal qualitative similarities in the thermodynamic landscapes for both riboswitches in which progression from the open, lysine-unbound state to both transition states (‡) and closed, lysine-bound conformations is enthalpically favored yet entropically penalized, with comparisons of enthalpic and entropic contributions extrapolated to a common [K+] = 100 mM in quantitative agreement. Finally, temperature-dependent Eyring analysis reveals the TMA and BSU riboswitches to have remarkably similar folding/unfolding rate constants when extrapolated to their respective (40 and 80°C) environmental temperatures. Such behavior suggests a shared strategy for ligand binding and aptamer conformational change in the two riboswitches, based on thermodynamic adaptations in number of G-C basepairs and/or modifications in tertiary structure that stabilize the ligand-unbound conformation to achieve biocompetence under both hyperthermophilic and mesothermophilic conditions.

中文翻译：

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枯草芽孢杆菌与马氏毛滴虫赖氨酸核糖开关中极端温度的热力学补偿


T. maritima 和 B. subtilis 是栖息在明显不同的热环境（∼80°C 和 ∼40°C）中的细菌，但采用相似的赖氨酸核糖开关来帮助参与氨基酸合成和运输的基因的转录调控。尽管 G-C 碱基对频率和一级序列存在显着差异，但每个核糖开关的适配子部分在三级结构上具有惊人的相似性，具有几个保守的基序和长程相互作用。为了探索极端热环境中的遗传适应，我们通过单分子荧光共振能量转移分析比较了 T. maritima 和 B. subtilis 赖氨酸核糖开关的动力学和热力学行为。动力学研究表明，核糖开关折叠率随着赖氨酸浓度的增加而增加，而去折叠率与赖氨酸无关。这表明两个核糖开关都通过诱导拟合（“结合后折叠”）机制结合赖氨酸，赖氨酸结合必然在构象变化之前发生。温度依赖性 van't Hoff 研究揭示了两种核糖开关的热力学景观的定性相似性，其中从开放、赖氨酸未结合状态进展到过渡态 （‡） 和闭合、赖氨酸结合的构象在焓上受到青睐，但在熵上受到惩罚，焓和熵贡献的比较外推到一个共同的 [K+] = 100 mM，在定量上一致。最后，温度依赖性 Eyring 分析表明，当外推到各自的环境温度（40°C 和 80°C）时，TMA 和 BSU 核糖开关具有非常相似的折叠/展开速率常数。 这种行为表明两个核糖开关中配体结合和适配体构象变化的共享策略，基于 G-C 碱基对数量的热力学适应和/或三级结构的修饰，这些修饰稳定配体未结合的构象，以在超嗜热和嗜热条件下实现生物能力。