Structural Analysis of the Multisynthetase Complex

多合成酶复合物的结构分析

• 批准号: 0090539
• 负责人: Michael Hebert
• 金额: --
• 依托单位: University of Mississippi Medical Center
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0090539&HistoricalAwards=false
• 关键词: Structural; Analysis; Multisynthetase; Complex

0090539NorcumAminoacyl-tRNA synthetases control fidelity of protein biosynthesis by accurate pairing of amino acids and transfer RNAs. Nine of these enzymes and three auxiliary proteins are isolated from multicellular eukaryotes as a macromolecular complex. The major biological function of the particle is likely organization of the translational machinery. One of its constituents is the precursor of a cytokine released during apoptosis and so the multisynthetase complex may also link biological signaling, programmed cell death and inflammation. This project will test and refine the three domain model of the rabbit reticulocyte multisynthetase complex. Its three-dimensional structure will be calculated from electron microscopic images of frozen, unstained samples. The reference volume obtained by random conical reconstruction methods will be refined by projection mapping. Toward the overall goal of mapping the full protein topography within the particle, this project will focus on a subset selected to provide a broad test of the working model. Using natural tRNAs or in vitro transcripts of synthetic tRNA genes as specific probes, at least one enzyme within each domain will be located. Work will begin with the arginyl-, leucyl- and glutaminyl-tRNA synthetases. Electron microscopy will provide general protein locations, which will be refined by difference mapping of control and labeled structures. Probes will be visualized directly or by combination of chemical labeling with reporter molecules. So that positions within at least one domain are confirmed and oriented, electron microscopy will be combined with fluorescence resonance energy transfer measurement of distances between pairs of bound fluorophore-labeled tRNAs. Work will begin with the "base", which contains leucyl-, isoleucyl- and bifunctional glutamyl-/prolyl-tRNA synthetases. This project's results are fundamental to understanding eukaryotic protein biosynthesis at the molecular level and to elucidation of principles underlying intracellular organization. The information obtained will also provide information about differences between prokaryotic and eukaryotic protein biosynthesis, a mechanism of autoimmunity, and a route to cell death.

NorcumAminoacyl-tRNA合成酶通过氨基酸和转移RNA的精确配对控制蛋白质生物合成的保真度。 这些酶中的九种和三种辅助蛋白质作为大分子复合物从多细胞真核生物中分离出来。 粒子的主要生物学功能可能是翻译机器的组织。 其成分之一是细胞凋亡过程中释放的细胞因子的前体，因此多合成酶复合物也可能将生物信号传导、程序性细胞死亡和炎症联系起来。 本计画将测试并改良兔网织红细胞多合成酶复合物的三结构域模型。 其三维结构将根据冷冻、未染色样品的电子显微镜图像计算。 通过随机圆锥重建方法获得的参考体积将通过投影映射进行细化。 为了实现绘制颗粒内完整蛋白质形貌的总体目标，该项目将重点关注选定的一个子集，以提供对工作模型的广泛测试。 使用天然tRNA或合成tRNA基因的体外转录物作为特异性探针，将定位每个结构域内的至少一种酶。 工作将开始与乙酰基-，亮氨酰-和乙酰氨基-tRNA合成酶。 电子显微镜将提供一般蛋白质的位置，这将是完善的控制和标记结构的差异映射。 探针将直接可视化或通过化学标记与报告分子的组合来可视化。 为了确认和定向至少一个结构域内的位置，电子显微镜将与结合的荧光团标记的tRNA对之间的距离的荧光共振能量转移测量相结合。 工作将开始与"基地"，其中包含亮氨酰-，异亮氨酰-和双功能谷氨酰-/脯氨酰-tRNA合成酶。 该项目的研究结果对于在分子水平上理解真核生物蛋白质的生物合成和阐明细胞内组织的基本原理具有重要意义。 所获得的信息还将提供有关原核和真核蛋白质生物合成之间的差异、自身免疫机制和细胞死亡途径的信息。