Structure of Eukaryotic Translation Elongation Factor 1

近年来，一系列可溶性翻译因子和核糖体本身的结构揭示了原核生物蛋白质合成的结构基础。对真核生物蛋白质合成过程中更为复杂和高度调控的步骤的类似理解才刚刚开始。本项目的目的是了解真核翻译伸长因子1A （eEF1A）的功能和调控的结构基础。eEF1A是一种原型g蛋白，不仅具有翻译功能，还具有tRNA输出、病毒复制和细胞骨架组织等功能。因此，很明显，细胞需要调节eEF1A的活性或水平来维持正常的细胞生长。鸟嘌呤核苷酸交换因子eEF1B对活性eEF1A的再生作用由我们的酿酒酵母eEF1A与eef1α亚基的催化片段络合的结构揭示。我们建议用1)GDP或GTP类似物GDPNP合成eEF1A结构；2)氨基酰trna （aa-tRNA）和GDPNP； 3)完整的鸟嘌呤核苷酸交换因子eEF1B和4)肌动蛋白。作为一种g蛋白，eEF1A根据是否结合GDP或GTP在活性和非活性形式之间切换。因此，了解两种形式之间的结构转换将有助于阐明与核糖体和aa-tRNA结合的调节。GTP形式结合aa-tRNA，该复合物的结构将阐明该复合物的整体三级结构，该复合物结合核糖体a位点并感知适当的密码子-反密码子相互作用。整个eEF1AB复合物的结构将有助于确定eEF1Bgamma亚基的功能，这是一种在所有真核生物中高度保守的蛋白质，但最近才涉及到可能调节翻译准确性和应激反应。最后，越来越多的证据表明，翻译装置的其他功能，特别是eEF1A结合和捆绑肌动蛋白的能力，使我们扩展了分析，以了解这种关联的结构基础。在酿酒酵母中，获得的eEF1A结构信息可用于功能和调控关键残基的分子遗传学分析。

Recently, the structural basis of protein synthesis in prokaryotes has been illuminated by a series of structures of the soluble translation factors and the ribosome itself. A similar understanding of the more complicated and highly regulated steps of eukaryotic protein synthesis is just beginning. The goal of this project is to understand the structural basis for the function and regulation of the eukaryotic translation Elongation Factor 1A (eEF1A). eEF1A is a prototypic G-protein that performs functions not only in translation but also tRNA export, viral replication and cytoskeletal organization. Thus, it is clear that the cell needs to modulate the activity or levels of eEF1A for normal cellular growth. The regeneration of active eEF1A by the guanine nucleotide exchange factor eEF1B is illuminated by our structure of Saccharomyces cerevisiae eEF1A complexed with the catalytic fragment of the eEF1Balpha subunit. We propose to produce structures of eEF1A in complex with 1) GDP or the GTP analogue GDPNP 2) aminoacyl-tRNA (aa-tRNA) and GDPNP, 3) the complete guanine nucleotide exchange factor eEF1B and 4) actin. As a G-protein, eEF1A switches between active and inactive forms The based on whether GDP or GTP is bound. Hence, understand the structural switch between the forms will help elucidate the regulation of binding to the ribosome and aa-tRNA. The GTP form binds aa-tRNA, and the structure of this complex will illuminate the overall tertiary structure of the complex that binds the ribosomal A-site and senses a proper codon-anticodon interaction. The structure of the entire eEF1AB complex will help determine the function of the eEF1Bgamma subunit, a protein highly conserved in all eucaryotes but only recently implicated in perhaps modulating translational accuracy and the stress response. Lastly, the growing evidence of alternative functions of the translational apparatus, and in particular the ability of eEF1A to bind and bundle actin, leads us to expand out analysis to understand the structural basis of this association. With S. cerevisiae, the structural information obtained on eEF1A can be utilized for molecular genetic analysis of the critical residues for function and regulation.