Structural Dynamics of Protein-RNA Complexes

描述（由申请人提供）：I组内含子是RNA基因中的非编码区，能够通过自剪接机制切除。内含子必须折叠成正确的天然结构以催化反应，尽管存在热力学和动力学挑战。来自粗糙脉孢菌线粒体RNA的I组内含子中的两个依赖于蛋白质辅因子CYT-18折叠成用于自我剪接的正确结构。第二种蛋白质CYT-19加速了这些蛋白质结合内含子的剪接速率。为了研究粗糙新孢子菌中多蛋白-RNA相互作用，将使用单分子荧光检测来自内含子的核酶。通过RNA的位点特异性染料标记，将监测特定结构域的结构动力学和单个分子的单个三级相互作用。与催化活性构象相关的结构转换将遵循蛋白质-RNA复合物。这项工作将有助于剖析蛋白质在弥补RNA折叠缺陷方面的进化作用。在这个模型系统中探索RNA-蛋白质相互作用的技术可能有助于为未来探索更复杂的健康相关系统的方法提供基础。

DESCRIPTION (provided by applicant): Group I introns are non-coding regions in RNA genes that are capable of excision through a self-splicing mechanism. The introns must fold to the correct native structure to catalyze the reaction despite thermodynamic and kinetic challenges. Two of the group I introns from the Neurospora crassa mitochondrial RNA are dependent on a protein cofactor, CYT-18, to fold to the correct structure for self-splicing. A second protein, CYT-19, accelerates the splicing rate for these protein-bound introns. To investigate the multiprotein-RNA interactions in Neuospora crassa, ribozymes derived from the introns will be examined using single molecule fluorescence. Through site-specific dye-labeling of the RNA, the structural dynamics of specific domains and single tertiary interactions for individual molecules will be monitored. Structural transitions associated with the catalytically active conformation will be followed for the protein-RNA complexes. The work will help to dissect the evolutionary role of proteins in compensating for deficiencies in RNA folding. The techniques developed in exploring the RNA-protein interactions in this model system may aid in providing the basis for future methodologies to probe more complicated health-related systems.