Protein Roles in Group I Intron RNA Folding: The Tyrosyl-tRNA Synthetase CYT-18 Stabilizes the Native State Relative to a Long-Lived Misfolded Structure without Compromising Folding Kinetics
Protein Roles in Group I Intron RNA Folding: The Tyrosyl-tRNA Synthetase CYT-18 Stabilizes the Native State Relative to a Long-Lived Misfolded Structure without Compromising Folding Kinetics
复制标题
DOI：
10.1016/j.jmb.2009.11.009
复制
发表时间：
2010-01-22
影响因子：
5.6
通讯作者：
Russell, Rick
中科院分区：
文献类型：
--
作者：
Chadee, Amanda B.;Bhaskaran, Hari;Russell, Rick
粗神经孢子虫CYT-18蛋白是一种线粒体酪氨酸- trna合成酶，通过稳定保守核心的功能结构，促进I族内含子rna的自剪接。CYT-18与常见外周元件P5abc沿同一表面结合核心，表明CYT-18可以在功能上取代P5abc。除了稳定结构外，P5abc还稳定了四膜虫I组内含子的天然构象，相对于全局相似的错误折叠构象，P5abc在核心内只有局部差异，并且在平衡状态下被缺乏P5abc的核酶变体（E-Delta P5abc）大量填充。在这里，我们证明了CYT-18特异性地促进了这种错误折叠构象的原生I族内含子核心的形成。催化活性分析表明，CYT-18将E-Delta P5abc的平衡向天然状态转移了至少35倍，结合分析表明其作用更大。因此，与P5abc类似，CYT-18优先识别原生核，尽管错误折叠的核具有全局相似性，尽管形成了粗略相似的配合物，如硫酸二甲酯足迹所示。有趣的是，CYT-18和P5abc对折叠动力学的影响不同。P5abc通过形成在重折叠过程中必须断裂的外周接触来抑制错误折叠构象的重折叠，而CYT-18没有表现出类似的抑制作用，很可能是因为它在更大程度上依赖于与核心的直接相互作用。尽管CYT-18在体内不会遇到这种RNA，但我们的研究结果表明，相对于类似的错误折叠中间物，CYT-18可以稳定其同源I族内含子。通过特异性识别天然结构特征，CYT-18也可能与早期折叠中间体相互作用，以避免RNA错误折叠或在它们形成时捕获天然接触。更一般地说，我们的结果强调了蛋白质辅助因子稳定功能性RNA结构的能力，而不会在RNA折叠动力学中产生相关成本。2009爱思唯尔有限公司版权所有。
The Neurospora crassa CYT-18 protein is a mitochondrial tyrosyl-tRNA synthetase that also promotes self-splicing of group I intron RNAs by stabilizing the functional structure in the conserved core. CYT-18 binds the core along the same surface as a common peripheral element, P5abc, suggesting that CYT-18 can replace P5abc functionally. In addition to stabilizing structure generally, P5abc stabilizes the native conformation of the Tetrahymena group I intron relative to a globally similar misfolded conformation that has only local differences within the core and is populated significantly at equilibrium by a ribozyme variant lacking P5abc (E-Delta P5abc). Here, we show that CYT-18 specifically promotes formation of the native group I intron core from this misfolded conformation. Catalytic activity assays demonstrate that CYT-18 shifts the equilibrium of E-Delta P5abc toward the native state by at least 35-fold, and binding assays suggest an even larger effect. Thus, similar to P5abc, CYT-18 preferentially recognizes the native core, despite the global similarity of the misfolded core and despite forming crudely similar complexes, as revealed by dimethyl sulfate footprinting. Interestingly, the effects of CYT-18 and P5abc on folding kinetics differ. Whereas P5abc inhibits refolding of the misfolded conformation by forming peripheral contacts that must break during refolding, CYT-18 does not display analogous inhibition, most likely because it relies to a greater extent on direct interactions with the core. Although CYT-18 does not encounter this RNA in vivo, our results suggest that it stabilizes its cognate group I introns relative to analogous misfolded intermediates. By specifically recognizing native structural features, CYT-18 may also interact with earlier folding intermediates to avoid RNA misfolding or to trap native contacts as they form. More generally, our results highlight the ability of a protein cofactor to stabilize a functional RNA structure specifically without incurring associated costs in RNA folding kinetics. (C) 2009 Elsevier Ltd. All rights reserved.