CAREER: Structural Studies of Ski2-like RNA Helicases

智力优势。 RNA监视和衰变途径对于维持适当的细胞功能和活力至关重要。这些RNA降解途径中的许多由Ski 2样RNA解旋酶介导，其占所有RNA解旋酶的10%。由于缺乏详细的结构和生物化学数据，对这些解旋酶如何识别和确保RNA底物适当衰变的完整机制理解相当有限。该项目的目标是通过晶体结构测定阐明这类重要酶的分子细节，并得到生化和生物分析的支持。该项目的一个重要焦点是Ski 2样Mtr 4蛋白，这是一种高度保守和必需的真核蛋白，在核RNA加工和衰变中起着核心作用。Mtr 4是核糖体RNA生物合成所需的，并且还靶向广泛的RNA以作为TRAMP（Trf 4-Air2-Mtr 4聚腺苷酸化）复合物的一部分进行降解。第二个靶标是Ski 2，Mtr 4的胞质对应物，其介导mRNA转录物的降解。该项目的具体组成部分包括：确定高分辨率晶体结构;鉴定重要的蛋白质-RNA相互作用;以及描述Ski 2样解旋酶所特有的新型拱形结构域。 该项目的更广泛的影响包括对结构生物学的本科生和研究生进行广泛的培训。结构生物学的一门新课程已经开发出来，它提供了蛋白质结构测定的严格实践训练。部门高级生物化学实验室课程已经重新设计，以适应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将进入结晶试验管道的单个蛋白质表达和纯化项目。高中生将参加夏季研究体验，介绍结晶和结构测定的基本概念。这种广泛的教育方法将广泛接触和直接进入结构生物学的迅速扩大的领域，这在历史上是不太容易接近地理偏远/较小的机构。在一个较小的大学校园中成功实施了一个新的结构生物学项目，该项目具有进行高端晶体学研究的全部能力，提供了一个可以在类似环境中复制的模板。该项目由分子和细胞生物科学部的基因和基因组系统以及刺激竞争性研究的实验项目共同资助。

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.