A thermodynamic framework and cooperativity in the tertiary folding of a Mg2+-dependent ribozyme
A thermodynamic framework and cooperativity in the tertiary folding of a Mg2+-dependent ribozyme
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DOI：
10.1021/bi991700n
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发表时间：
1999-12-21
期刊：
影响因子：
2.9
通讯作者：
Sosnick, TR
中科院分区：
文献类型：
--
作者：
Fang, XW;Pan, T;Sosnick, TR
利用圆二色谱和荧光光谱、羟基自由基保护和催化活性分析了枯草芽孢杆菌RNase P RNA催化结构域的折叠热力学。这种255个核苷酸的核酶的折叠可以用三种填充的种类来描述：未折叠（U）、中间体（I）和天然（N）状态。U到I的转变主要涉及二级结构的形成，而I到N的转变主要是由三级结构的形成。I-N跃迁是高度合作的，如这里应用的四个探针的重合所示。使用两种等温方法来确定N态相对于I态在10 ℃和37 ℃下的稳定性。第一种方法测量Mg 2+诱导的折叠的程度没有尿素或在恒定的尿素浓度。第二种方法测量在恒定Mg 2+浓度下尿素诱导的展开程度。通过应用协同结合分析，通过两种方法确定的Mg 2+过渡中点（K-Mg）、希尔常数（n）和尿素依赖性表面埋藏参数（In值）是相同的，表明它们报告了相同的可逆折叠事件。从这些结果可以得出三个结论。(i)Mg ~（2+）依赖的RNA三级结构的折叠自由能可以用K-Mg参数和n参数来描述。(ii)这种三级RNA结构的希尔常数可能代表了在I到N跃迁中结合的Mg 2+离子的微分数。(iii)在生理条件下，这种大核酶的稳定性与小球状蛋白相似。
The folding thermodynamics of the catalytic domain from the Bacillus subtilis RNase P RNA is analyzed using circular dichroism and fluorescence spectroscopies, hydroxyl radical protection, and catalytic activity. Folding of this 255-nucleotide ribozyme can be described with three populated species: unfolded (U), intermediate (I), and native (N) states. The U-to-I transition primarily involves secondary structure formation, whereas the I-to-N transition is dominated by tertiary structure formation. The I-to-N transition is highly cooperative as indicated by the coincidence of the four probes applied here. Two isothermal methods are used to determine the stability of the N state relative to the I state at 10 and 37 degrees C. The first method measures the extent of Mg2+-induced folding without urea or at constant urea concentrations. The second method measures the extent of urea-induced unfolding at constant Mg2+ concentrations. Via application of a cooperative binding analysis, the Mg2+ transition midpoint (K-Mg), the Hill constant (n), and the urea-dependent surface burial parameter (In value) determined by both methods are identical, indicating that they report the same, reversible folding event. Three conclusions can be drawn from these results. (i) The folding free energy of a Mg2+-dependent tertiary RNA structure can be described by the K-Mg and n parameters according to a cooperative Mg2+ binding model. (ii) The Hill constant for this tertiary RNA structure probably represents the differential number of Mg2+ ions bound in the I-to-N transition. (iii) Under physiological conditions, the stability of this large ribozyme is similar to that of small globular proteins.