代谢组削弱 RNA 热力学稳定性并增强 RNA 化学稳定性,Biochemistry

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代谢组削弱 RNA 热力学稳定性并增强 RNA 化学稳定性
Biochemistry ( IF 2.9 ) Pub Date : 2022-10-28 , DOI: 10.1021/acs.biochem.2c00488
Jacob P Sieg _{1,

2} , Lauren N McKinley _{1,

2} , Melanie J Huot _{3,

4} , Neela H Yennawar ₅ , Philip C Bevilacqua _{1,

2,

3}

Affiliation

我们在模拟大肠杆菌细胞质的条件下检查了 RNA、代谢组和二价 Mg ²⁺之间相互作用的复杂网络。我们确定了前 15 种大肠杆菌代谢物的 Mg ²⁺结合常数，按生理 pH 浓度和单价离子浓度计算，占代谢组的 80%。这些数据用于模拟体内大肠杆菌条件的人工细胞质的开发，我们将其称为“Eco80”。我们根据经验确定， Eco80 中的大肠杆菌代谢物混合物在生物学相关的游离 Mg ²⁺范围（约 0.5 至 3 mM Mg ²⁺^）中近似于对 Mg 2+ 的单位点结合行为，使用 Mg ²⁺敏感荧光染料。研究了 Eco80 条件对 RNA 热力学稳定性、化学稳定性、结构和催化作用的影响。我们发现 Eco80 条件会对 RNA 的热力学和化学稳定性产生相反的影响。特别是，RNA螺旋的热力学稳定性减弱了0.69 ± 0.12 kcal/mol，而化学稳定性则增强了~2倍，这可以通过弱和强Mg 2+ 代谢物之间^Mg 2+^的形态来理解。 Eco80 中的复合体。总体而言，Eco80 的使用反映了 RNA体内功能，并增强了 RNA 机制研究的生物学相关性。

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The Metabolome Weakens RNA Thermodynamic Stability and Strengthens RNA Chemical Stability

We examined the complex network of interactions among RNA, the metabolome, and divalent Mg²⁺ under conditions that mimic the Escherichia coli cytoplasm. We determined Mg²⁺ binding constants for the top 15 E. coli metabolites, comprising 80% of the metabolome by concentration at physiological pH and monovalent ion concentrations. These data were used to inform the development of an artificial cytoplasm that mimics in vivoE. coli conditions, which we term “Eco80”. We empirically determined that the mixture of E. coli metabolites in Eco80 approximated single-site binding behavior toward Mg²⁺ in the biologically relevant free Mg²⁺ range of ∼0.5 to 3 mM Mg²⁺, using a Mg²⁺-sensitive fluorescent dye. Effects of Eco80 conditions on the thermodynamic stability, chemical stability, structure, and catalysis of RNA were examined. We found that Eco80 conditions lead to opposing effects on the thermodynamic and chemical stabilities of RNA. In particular, the thermodynamic stability of RNA helices was weakened by 0.69 ± 0.12 kcal/mol, while the chemical stability was enhanced ∼2-fold, which can be understood using the speciation of Mg²⁺ between weak and strong Mg²⁺–metabolite complexes in Eco80. Overall, the use of Eco80 reflects RNA function in vivo and enhances the biological relevance of mechanistic studies of RNA.

更新日期：2022-10-28

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