Structural Analysis of the Multisynthetase Complex

去甲氨基酰基- trna合成酶通过氨基酸和转移rna的精确配对来控制蛋白质生物合成的保真度。其中9种酶和3种辅助蛋白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组织翻译机制。它的一种成分是细胞凋亡过程中释放的细胞因子的前体，因此多合成酶复合体也可能与生物信号、程序性细胞死亡和炎症有关。本项目将测试和完善兔网织细胞多合成酶复合物的三域模型。它的三维结构将从冷冻、未染色样品的电子显微镜图像中计算出来。随机圆锥重建方法得到的参考体将通过投影映射进行细化。为了实现在颗粒内绘制完整蛋白质地形的总体目标，该项目将重点关注选定的子集，以提供对工作模型的广泛测试。使用天然tRNA或人工合成tRNA基因的体外转录物作为特异性探针，每个结构域内至少有一种酶将被定位。工作将从精氨酸-、亮氨酸-和谷氨酰胺- trna合成酶开始。电子显微镜将提供一般的蛋白质位置，这将通过对照和标记结构的差异映射来改进。探针将直接可视化或结合化学标记与报告分子。因此，在至少一个区域内的位置被确认和定向，电子显微镜将与荧光共振能量转移测量结合的荧光团标记的trna对之间的距离相结合。工作将从“碱基”开始，它包含亮氨酸、异亮氨酸和双功能谷氨酰/脯氨酸- trna合成酶。该项目的结果是在分子水平上理解真核蛋白生物合成和阐明细胞内组织的基本原理的基础。获得的信息还将提供有关原核和真核蛋白质生物合成差异、自身免疫机制和细胞死亡途径的信息。

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.