Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, United States.
Biochemistry. 2011 Feb 8;50(5):864-74. doi: 10.1021/bi101467q. Epub 2011 Jan 11.
Structured RNAs encode native conformations that are more stable than the vast ensembles of alternative conformations, but how this specificity is evolved is incompletely understood. Here we show that a variant of the Tetrahymena group I intron ribozyme that was generated previously by in vitro selection for enhanced thermostability also displays modestly enhanced specificity against a stable misfolded structure that is globally similar to the native state, despite the absence of selective pressure to increase the energy gap between these structures. The enhanced specificity for native folding arises from mutations in two nucleotides that are close together in space in the native structure, and additional experiments show that these two mutations do not affect the stability of the misfolded conformation relative to the largely unstructured transition state ensemble for interconversion between the native and misfolded conformers. Thus, they selectively stabilize the native state, presumably by strengthening a local tertiary contact network that cannot form in the misfolded conformation. The stabilization is larger in the presence of the peripheral element P5abc, suggesting that cooperative tertiary structure formation plays a key role in the enhanced stability. The increased specificity in the absence of explicit selection suggests that the large energy gap in the wild-type RNA may have arisen analogously, a consequence of selective pressure for stability of the functional structure. More generally, the structural rigidity and intricate networks of contacts in structured RNAs may allow them to evolve substantial structural specificity without explicit negative selection, even against closely related alternative structures.
结构 RNA 编码的天然构象比大量的替代构象更稳定，但这种特异性是如何进化的还不完全清楚。在这里，我们展示了一种之前通过体外选择增强热稳定性产生的 Tetrahymena 组 I 内含子核酶变体，尽管没有选择性压力来增加这些结构之间的能量差距，但它对与天然状态全局相似的稳定错误折叠结构也表现出适度增强的特异性。对天然折叠的增强特异性来自于两个核苷酸的突变，这两个核苷酸在天然结构中空间上彼此靠近，并且额外的实验表明，这两个突变不会影响错误折叠构象相对于主要无结构的转换状态集之间的稳定性，这些构象之间的相互转化是形成天然和错误折叠构象的。因此，它们选择性地稳定天然状态，可能是通过加强局部三级接触网络，该网络不能在错误折叠构象中形成。在外周元件 P5abc 的存在下，稳定性更大，这表明协同的三级结构形成在增强稳定性中起着关键作用。在没有明确选择的情况下特异性增加表明，野生型 RNA 中的大能量差距可能以类似的方式出现，这是功能结构稳定性的选择性压力的结果。更一般地说，结构 RNA 中的结构刚性和复杂的接触网络可能使它们能够在没有明确负选择的情况下进化出相当大的结构特异性，即使是针对密切相关的替代结构。
