Rook M S, Treiber D K, Williamson J R
Department of Molecular Biology and the Skaggs Institute for Chemistry and Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037, USA.
J Mol Biol. 1998 Aug 28;281(4):609-20. doi: 10.1006/jmbi.1998.1960.
A model for the kinetic folding pathway of the Tetrahymena ribozyme has been proposed where the two main structural domains, P4-P6 and P3-P7, form in a hierarchical manner with P4-P6 forming first and P3-P7 folding on the minute timescale. Recent studies in our laboratory identified a set of mutations that accelerate P3-P7 formation, and all of these mutations appear to destabilize a native-like kinetic trap. To better understand the microscopic details of this slow step in the Tetrahymena ribozyme folding pathway, we have used a previously developed kinetic oligonucleotide hybridization assay to characterize the folding of several fast folding mutants. A comparison of the temperature dependence of P3-P7 folding between the mutant and wild-type ribozymes demonstrates that a majority of the mutations act by decreasing the activation enthalpy required to reach the transition state and supports the existence of the native-like kinetic trap. In several mutant ribozymes, P3-P7 folds with biphasic kinetics, indicating that only a subpopulation of molecules can evade the kinetic barrier. The rate of folding of the wild-type increases in the presence of urea, while for the mutants urea merely shifts the distribution between the two folding populations. Small structural changes or changes in solvent can accelerate folding, but these changes lead to complex folding behavior, and do not give rise to rapid two-state folding transitions. These results support the recent view of folding as an ensemble of molecules traversing a rugged energy landscape to reach the lowest energy state.
有人提出了四膜虫核酶动力学折叠途径的模型，其中两个主要结构域P4 - P6和P3 - P7以分层方式形成，P4 - P6先形成，P3 - P7在分钟时间尺度上折叠。我们实验室最近的研究鉴定出一组加速P3 - P7形成的突变，所有这些突变似乎都会破坏一种类似天然的动力学陷阱。为了更好地理解四膜虫核酶折叠途径中这一慢步骤的微观细节，我们使用了先前开发的动力学寡核苷酸杂交测定法来表征几种快速折叠突变体的折叠情况。突变型和野生型核酶之间P3 - P7折叠的温度依赖性比较表明，大多数突变通过降低达到过渡态所需的活化焓起作用，并支持类似天然的动力学陷阱的存在。在几种突变型核酶中，P3 - P7以双相动力学折叠，表明只有一部分分子能够避开动力学屏障。在尿素存在下，野生型的折叠速率增加，而对于突变体，尿素只是改变了两个折叠群体之间的分布。小的结构变化或溶剂变化可以加速折叠，但这些变化会导致复杂的折叠行为，不会产生快速的两态折叠转变。这些结果支持了最近关于折叠是分子集合体穿越崎岖能量景观以达到最低能量状态的观点。
