JILA, University of Colorado Boulder and National Institute of Standards and Technology, Boulder, Colorado; Department of Chemistry, University of Colorado Boulder, Boulder, Colorado.
JILA, University of Colorado Boulder and National Institute of Standards and Technology, Boulder, Colorado; Department of Chemistry, University of Colorado Boulder, Boulder, Colorado; Department of Physics, University of Colorado Boulder, Boulder, Colorado.
Biophys J. 2024 Oct 1;123(19):3331-3345. doi: 10.1016/j.bpj.2024.07.039. Epub 2024 Jul 31.
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.
海洋栖热菌（T. maritima）和枯草芽孢杆菌（B. subtilis）是两种栖息在截然不同的热环境中的细菌，分别约为 80°C 和 40°C。然而，它们采用相似的赖氨酸核糖开关来帮助调节参与氨基酸合成和转运的基因的转录。尽管在 G-C 碱基对频率和一级序列上存在显著差异，但每个核糖开关的适体部分在三级结构上具有惊人的相似性，存在几个保守基序和长程相互作用。为了探索极端热环境中的遗传适应性，我们通过单分子荧光共振能量转移分析比较了海洋栖热菌和枯草芽孢杆菌赖氨酸核糖开关的动力学和热力学行为。动力学研究表明，随着赖氨酸浓度的增加，核糖开关折叠速率增加，而解折叠速率与赖氨酸浓度无关。这表明这两种核糖开关都通过诱导契合（“结合后折叠”）机制结合赖氨酸，赖氨酸结合必然先于构象变化。温度依赖的范特霍夫研究揭示了这两种核糖开关在热力学景观上存在定性相似性，其中从开放、无赖氨酸结合的状态到两个过渡态（‡）和闭合、赖氨酸结合的构象的转变在焓上是有利的，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 时在定量上是一致的。最后，温度依赖的艾林分析表明，当外推到各自的（40°C 和 80°C）环境温度时，TMA 和 BSU 核糖开关的折叠/解折叠速率常数非常相似。这种行为表明，在这两个核糖开关中，配体结合和适体构象变化存在一种共享策略，这是基于 G-C 碱基对数量的热力学适应和/或三级结构的修饰，这些修饰稳定了配体未结合的构象，以在高温和中温条件下实现生物相容性。
