Applicability of urea in the thermodynamic analysis of secondary and tertiary RNA folding
Applicability of urea in the thermodynamic analysis of secondary and tertiary RNA folding
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DOI：
10.1021/bi991699s
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发表时间：
1999-12-21
期刊：
影响因子：
2.9
通讯作者：
Pan, T
中科院分区：
文献类型：
--
作者：
Shelton, VM;Sosnick, TR;Pan, T
在等温条件下，用光学光谱和化学修饰研究了一系列自互补RNA双链和未修饰酵母tRNA(Phe)的平衡折叠随尿素和镁离子浓度的变化。通过应用蛋白质折叠的标准方法，确定了折叠自由能及其与尿素浓度的依赖关系m值。从尿素滴定得到的RNA双链的自由能与从热熔融研究中计算的自由能很好地一致[Freier，S.I.，et al.(1986)Proc.娜塔莉。阿卡德。SCI。美国第83,9373号]。M值与RNA双链的长度相关，对离子条件和温度不敏感。未经修饰的酵母tRNAPhe的折叠可由两个依赖于镁离子的转变来描述，第二个转变对应于羟基自由基保护和部分核酸酶消化所证实的天然三级结构的形成。这两个转变对尿素都很敏感，其m值分别为0.94和1.70千卡摩尔(-1)M~(-1)。虽然RNA尿素变性的确切化学基础尚不确定，但双链和tRNA(Phe)的Z值与折叠转变中埋藏的表面积成正比。这种相称性。0.099卡·摩尔(-1)M~(-1)埃(-2)，与蛋白质，0.11卡·摩尔(-1)M~(-1)埃(-2)非常相似[Myers，J.，Pace，N.，and Scholtz，M.(1995年)蛋白质科学。4,2138]。这些结果表明，尿素滴定可以用来测量RNA折叠转变的自由能和大小。
The equilibrium folding of a series of self-complementary RNA duplexes and the unmodified yeast tRNA(Phe) is studied as a function of urea and Mg2+ concentration with optical spectroscopies and chemical modification under isothermal conditions. Via application of standard methodologies from protein folding, the folding free energy and its dependence on urea concentration, the m value, are determined. The free energies of the RNA duplexes obtained from the urea titrations are in good agreement with those calculated from thermal melting studies [Freier, S. I., et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9373]. The m value correlates with the length of the RNA duplex and is not sensitive to ionic conditions and temperature. The folding of the unmodified yeast tRNAPhe,,, be described by two Mg2+-dependent transitions, the second of which corresponds to the formation of the native tertiary structure as confirmed by hydroxyl radical protection and partial nuclease digestion. Both transitions are sensitive to urea and have m values of 0.94 and 1.70 kcal mol(-1) M-1, respectively. Although the precise chemical basis of urea denaturation of RNA is uncertain, the nz values for the duplexes and tRNA(Phe) are proportional to the amount of the surface area buried in the folding transition. This proportionality. 0.099 cal mol(-1) M-1 Angstrom(-2), is very similar to that observed for proteins, 0.11 cal mol(-1) M-1 Angstrom(-2) [Myers, J., Pace, N., and Scholtz, M. (1995) Protein Sci. 4, 2138]. These results indicate that urea titration can be used to measure both the free energy and the magnitude of an RNA folding transition.