MECHANISTIC STUDIES OF THE PUTATIVE RNA HELICASE DBPA

假定的 RNA 解旋酶 DBPA 的机制研究

• 批准号: 2862084
• 负责人: KEVIN J POLACH
• 金额: $ 3.03万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2862084
• 关键词: RNA; RNA binding protein; adenosine triphosphate; adenosinetriphosphatase; analytical ultracentrifugation; calorimetry; conformation; crosslink; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate complex; fluorescence resonance energy transfer; helicase; hydrolysis; ribosomal RNA; stoichiometry; thermodynamics

The long-term objective of this project is to establish the molecular functions of DEAD box proteins. These RNA dependent ATPases are critical to most cellular processes involving RNA including translational initiation, mRNA splicing, and assembly of RNA-protein complexes such as the ribosome. Th model system for these studies will be the E. coli protein DbpA, which is unique among DEAD box proteins in that it displays high affinity and specificity for 23S rRNA. The specific aims of this project are 1) a thorough biophysical characterization of the specific enzyme-substrate complex and 2) a detailed understanding of the molecular consequences of ATP hydrolysis in terms of conformational changes in DbpA and the RNA substrate. Size exclusion chromatography and analytical ultracentrifugation will provide information on stoichiometries and RNA-protein crosslinking will identify domains important to substrate specificity. Questions of functionality will be addressed through in vitro helicase assays, fluorescence energy transfer experiments, and differential scanning calorimetry. Quantitative analysis of the enzymology of DbpA will lead to new insights for the mode of action of this important class of proteins.

该项目的长期目标是建立DEAD盒蛋白的分子功能。 这些RNA依赖性ATP酶对大多数涉及RNA的细胞过程至关重要，包括翻译起始、mRNA剪接和RNA-蛋白质复合物如核糖体的组装。 这些研究的模型系统将是E.在DEAD box蛋白中，DbpA是一种独特的对23 SrRNA具有高亲和力和特异性的蛋白。 该项目的具体目标是：1）对特定酶-底物复合物进行彻底的生物物理表征; 2）详细了解ATP水解在DbpA和RNA底物构象变化方面的分子后果。 尺寸排阻色谱和分析超离心将提供化学计量的信息和RNA-蛋白质交联将确定重要的底物特异性的结构域。 功能性的问题将通过体外解旋酶测定，荧光能量转移实验和差示扫描量热法来解决。 DbpA酶学的定量分析将导致这类重要蛋白质的作用模式的新见解。