Structural Analysis of the Multisynthetase Complex

正常氨基酰-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。其中9种酶和3种辅助蛋白是以大分子复合体的形式从多细胞真核生物中分离出来的。粒子的主要生物学功能可能是翻译机器的组织。它的一个成分是在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将生物信号、细胞程序性死亡和炎症联系在一起。本项目将测试和提炼兔网织红细胞多合酶复合体的三个结构域模型。它的三维结构将从冷冻的未染色样本的电子显微镜图像中计算出来。通过投影映射对随机圆锥重建方法得到的参考体进行精化。朝着绘制粒子内完整蛋白质拓扑图的总体目标，该项目将重点放在选定的子集上，以提供工作模型的广泛测试。使用天然tRNAs或人工合成tRNA基因的体外转录本作为特定探针，将在每个结构域中至少定位一个酶。工作将从精氨酸基、亮氨基和谷氨酰tRNA合成酶开始。电子显微镜将提供大致的蛋白质位置，这些位置将通过对照和标记结构的差异图谱进行细化。探针将直接或通过化学标记与报告分子相结合来可视化。为了确认和定位至少一个结构域中的位置，电子显微镜将与荧光共振能量转移测量结合在一起，测量结合的荧光团标记的tRNA对之间的距离。工作将从“碱”开始，它包含亮氨酰基、异亮氨基和双功能谷氨酰基/脯氨基-tRNA合成酶。该项目的结果对于在分子水平上理解真核蛋白质的生物合成和阐明潜在的细胞内组织原理是基本的。所获得的信息还将提供有关原核生物和真核生物蛋白质生物合成的差异、自身免疫的机制以及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.