Molecular Basis of Group II Intron Domain 5 Catalytic Function

本研究员的长期研究目标是使用分子生物学、生物化学和结构生物学的综合方法来了解剪接催化机制的结构基础。目前的项目旨在了解RNA相互作用如何使II组内含子内的催化装置的组装成核以进行剪接。因为结构域5（D5）是这种内含子RNA酶（核酶）的遗传学上最保守的区域，并且是由II组内含子或其衍生物催化的任何反应所绝对需要的，所以本项目集中于D5。以下两个具体目标旨在充分阐明D5如何通过特定的RNA-RNA和RNA-离子相互作用招募催化机制的组分，以形成必要的活性位点结构。目的1-确定II组内含子的催化必需的D5-PL的结构;目的2-检验D5-PL在结合结构域1-3上经历局部构象变化以建立催化活性位点几何形状的假设。这项研究有望为D5在II组内含子催化中的作用提供详细的分子理解。由此产生的更深入的了解内的第二组内含子的催化的性质可能会建议新的方法来开发第二组内含子作为潜在的核酶治疗。相应地，与本研究相关的教育活动旨在展示RNA分子在细胞代谢中发挥的多功能和多样性作用，该项目为RNA生物化学和NMR结构生物物理学的研究生和博士后的跨学科培训，辅导和指导提供了一个很好的途径。

The long-term research objective for this investigator is to use a combined approach of molecular biology, biochemistry, and structural biology to understand the structural basis for the catalytic mechanism of splicing. The current project is aimed at understanding how RNA interactions nucleate the assembly of the catalytic apparatus within the group II introns to undertake splicing. Because domain 5 (D5) is the most phylogenetically conserved region of this intron RNA enzyme (ribozyme) and is absolutely required for any reaction catalyzed by group II introns or its derivatives, this project focuses on D5. The following two specific aims are designed to fully illuminate how D5 recruits components of the catalytic machinery through specific RNA-RNA and RNA-ion interactions to form essential active-site structures. Aim 1-To determine the structure of the catalytically essential D5-PL of group II introns; Aim 2-To test the hypothesis that D5-PL undergoes localized conformational changes on binding domains 1-3 to set up the active site geometry for catalysis. This study is expected to provide detailed molecular understanding of the role of D5 in group II intron catalysis. The resulting deeper understanding of the nature of catalysis within the group II introns might suggest novel approaches to developing group II introns as potential ribozyme therapeutics. Correspondingly, the educational activities connected with this research are designed to demonstrate the versatile and diverse roles RNA molecules play in cellular metabolism, and the project provides an excellent avenue for the interdisciplinary training, tutoring and mentoring of graduate and postdoctoral students in RNA biochemistry and NMR structural biophysics.