CAREER: Structural Studies of Ski2-like RNA Helicases

智力上的功绩。RNA监测和衰变途径对于维持细胞的正常功能和活性是必不可少的。其中许多RNA衰变途径是由类Ski2 RNA解旋酶介导的，占所有RNA解旋酶的10%。由于缺乏详细的结构和生化数据，对这些解旋酶如何发挥作用以识别和确保RNA底物适当衰退的完整机械理解相当有限。这个项目的目标是通过晶体结构测定，并辅之以生化和生物学分析，阐明这类重要酶的分子细节。该项目的一个重要焦点是Ski2样Mtr4蛋白，这是一种高度保守的真核生物蛋白质，在核RNA的加工和衰退中发挥核心作用。MTR4是核糖体RNA生物发生所必需的，也是TRAMP(Trf4-Air2-MTR4多腺苷化)复合体的一部分，靶向广泛的RNA进行降解。第二个靶点是Ski2，它是mtr4的胞质对应物，介导mRNA转录产物的降解。该项目的具体组成部分包括：高分辨率晶体结构的确定；重要的蛋白质-RNA相互作用的确定；以及对Ski2类螺旋酶特有的新的ARCH结构域的表征。该项目的更广泛影响包括对本科生和研究生进行结构生物学方面的广泛培训。一门新的结构生物学课程已经开发出来，它提供了在蛋白质结构确定方面的严格实践培训。系高级生物化学实验室课程已重新设计，以适应一个新的和不断增长的生物化学专业。为了将基于研究的实验引入实验室课程，学生小组将被分配到单独的蛋白质表达和纯化项目，这些项目将进入结晶试验的管道。高中生将参加暑期研究体验，介绍结晶和结构确定的基本概念。这种广泛的教育方法将使人们广泛接触并直接进入迅速扩大的结构生物学领域，而从历史上看，地理位置偏远/规模较小的机构很难接触到这一领域。在一个较小的大学校园成功实施了一个新的结构生物学项目，具有进行高端结晶学研究的全部能力，提供了一个可以在类似环境中复制的模板。由分子和细胞生物科学系的基因和基因组系统以及刺激竞争性研究的实验计划共同资助。

Intellectual Merit. RNA surveillance and decay pathways are essential for maintaining proper cellular function and viability. Many of these RNA decay pathways are mediated by Ski2-like RNA helicases, which account for 10% of all RNA helicases. A complete mechanistic understanding of how these helicases function to recognize and ensure appropriate decay of RNA substrates is considerably limited due to a lack of detailed structural and biochemical data. The goal of this project is to elucidate the molecular details of this important class of enzymes through crystal structure determination, supported by biochemical and biological analyses. An important focus of this project is the Ski2-like Mtr4 protein, a highly conserved and essential eukaryotic protein that plays a central role in nuclear RNA processing and decay. Mtr4 is required for ribosomal RNA biogenesis, and also targets a wide array of RNAs for degradation as part of the TRAMP (Trf4-Air2-Mtr4 polyadenylation) complex. A second target is Ski2, the cytosolic counterpart of Mtr4, which mediates degradation of mRNA transcripts. The specific components of this project include: determination of high-resolution crystal structures; identification of important protein-RNA interactions; and characterization of a novel arch domain that is unique to Ski2-like helicases.Broader Impacts. The broader impacts of this project include extensive training of undergraduate and graduate students in structural biology. A new course in structural biology has been developed which provides rigorous hands-on training in protein structure determination. The departmental senior-level biochemistry laboratory course has been redesigned to accommodate a new and growing biochemistry major. In an effort to introduce research-based experiments into the laboratory course, student groups will be assigned individual protein expression and purification projects that will enter the pipeline for crystallization trials. High school students will participate in a summer research experience to introduce fundamental concepts of crystallization and structure determination. This broad educational approach will give widespread exposure and direct access to the rapidly expanding field of structural biology, which has historically been less accessible to geographically remote/smaller institutions. The successful implementation of a new program in structural biology at a smaller university campus, with the full capacity to perform high-end crystallographic research, provides a template that may be reproduced in similar environments.Co-funded by Genes and Genome Systems in the Division of Molecular and Cellular Biosciences and by the Experimental Program to Stimulate Competitive Research.