CAREER: Dissecting Inter-domain Communication in Methionyl-tRNA Synthetase

本项目的最终目标是解释蛋白质-RNA的构象动力学。为了有效的催化，来自大肠杆菌甲硫酰-tRNA合成酶(MetRS)反密码子结合域的信息必须在与酶的同源tRNA相互作用时被传递到远程活性部位。生化证据表明，这一信号事件需要改变RNA和蛋白质结构。这种长距离的构象变化在核糖体和剪接体等大分子机器中可能是非常常见的，因此具有基本的生物学意义，但它们实际上仍然没有被探索。适合进行详细、定量分析的MetRS模型系统将有助于弥补这一差距。突变、动力学和光谱方法将被用来研究MetRs结构域界面上的特定多肽成分如何促进域间通信。此外，还将使用结晶学来观察MetRs与其同源tRNA和底物类似物以及与未连接enyzme的络合物之间的结构差异。这项跨学科研究的结果将有助于对氨基酰-tRNA合成酶的域间通讯有一个新的理解，并有助于对具有远距离配体结合和催化活性部位的酶的信号机制的一般知识。广泛的影响：该项目将在维克森林大学进行生物、化学和物理接口的本科生和研究生教育。这位PI将她对科学研究的知识和热情带到当地公立学校正在进行的外展项目中。这些教师讲习班和学生充实活动的生物技术重点加强了地方努力，以激励和培训农村和城市高中毕业生从事中学后科学职业。这些努力既促进了PI在研究和教育方面的职业发展，也促进了社区着眼于经济增长的科学意识和培训。

This project's ultimate goal is to explain protein-RNA conformational dynamics.For efficient catalysis, information from the Escherichia coli methionyl-tRNA synthetase (MetRS) anticodon binding domain must be transmitted to a remote active site upon interaction with the enzyme's cognate tRNA. Biochemical evidence suggests this signaling event requires changes in both RNA and protein structure. Such long-range conformational changes are probably very common in macromolecular machines, such as ribosomes and spliceosomes, and, therefore, of fundamental biological importance, yet they remain virtually unexplored. The MetRS model system, amenable to detailed, quantitative analysis, will help to remedy this gap. Mutagenetic, kinetic, and spectroscopic approaches will be used to investigate how a specific peptide component at the domain interface of MetRS contributes to inter-domain communication. In addition, crystallography will be used to observe structural differences between MetRS in complex with its cognate tRNA and substrate analogs and with unliganded enyzme. Results obtained in this interdisciplinary study will contribute to a new understanding of inter-domain communication in aminoacyl-tRNA synthetases and to general knowledge about signaling mechanisms in enzymes with distant ligand-binding and catalytic active sites.Broader Impacts: The project will engage undergraduate and graduate education at the interface of biology, chemistry, and physics at Wake Forest University. The PI carries her knowledge of, and enthusiasm for, science research to ongoing outreach programs in local public schools. The biotechnology focus of these teacher workshops and student enrichment activities strengthens local efforts to motivate and to train rural and urban high school graduates to pursue postsecondary science careers. These efforts enhance both the PI's career development in research and education and the community's scientific awareness and training with an eye toward economic growth.