MECHANISTIC STUDIES OF THE PUTATIVE RNA HELICASE DBPA

该项目的长期目标是确定DEAD box蛋白的分子功能。这些依赖RNA的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-蛋白复合物（如核糖体）的组装。这些研究的模型系统将是大肠杆菌蛋白DbpA，它在DEAD box蛋白中是独特的，因为它对23S rRNA具有高亲和力和特异性。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水解对DbpA和RNA底物构象变化的分子后果。尺寸排除色谱和分析性超离心将提供化学计量学信息，rna -蛋白交联将识别对底物特异性重要的结构域。功能性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。对DbpA酶学的定量分析将为这类重要蛋白质的作用模式提供新的见解。

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.