Molecular Basis of Group II Intron Domain 5 Catalytic Function

该研究人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组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相互作用如何使第二类内含子内的催化装置组装成核，以进行剪接。由于结构域5(D5)是这种内含子RNA酶(核酶)在系统发育上最保守的区域，并且对于由第二组内含子或其衍生物催化的任何反应都是绝对必需的，因此本项目重点研究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械的组件，以形成必要的活性部位结构。目的1-确定第二组内含子催化必需的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中心几何结构的假设。这项研究有望为D5在第二组内含子催化中的作用提供详细的分子理解。由此产生的对第二组内含子内催化性质的深入了解可能会为开发第二组内含子作为潜在的核酶疗法提供新的方法。相应地，与这项研究相关的教育活动旨在展示RNA分子在细胞新陈代谢中扮演的多才多艺的角色，该项目为研究生和博士后在RNA生物化学和核磁共振结构生物物理学方面的跨学科培训、辅导和指导提供了一个极好的途径。

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.