Structure of Eukaryotic Translation Elongation Factor 1

近年来，原核生物蛋白质合成的结构基础已被一系列可溶性翻译因子和核糖体本身的结构所阐明。对真核生物蛋白质合成的更复杂和更严格调控步骤的类似理解才刚刚开始。本项目的目的是了解真核细胞翻译延伸因子1A(EEF1A)的功能和调控的结构基础。EEF1a是一种典型的G蛋白，不仅在翻译中发挥功能，而且在tRNA输出、病毒复制和细胞骨架组织中也发挥功能。因此，很明显，为了细胞的正常生长，细胞需要调节eEF1a的活性或水平。通过我们的酿酒酵母eEF1a与eEF1Balpha亚基催化片段的络合结构，说明了鸟核苷酸交换因子eEF1B对活性eEF1A的再生作用。我们建议在与1)GDP或GTP类似物GDPNP 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)肌动蛋白的复合体中产生eEF1A的结构。作为一种G蛋白，eEF1a根据GDP或GTP的结合情况在活性形式和非活性形式之间进行切换。因此，了解这些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形式与AA-tRNA结合，该复合体的结构将揭示与核糖体A位点结合的复合体的整体三级结构，并检测到适当的密码子-反密码子相互作用。整个eEF1AB复合体的结构将有助于确定eEF1BGamma亚基的功能，eEF1BGamma亚基是一种在所有真核生物中高度保守的蛋白质，但直到最近才被发现可能参与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机构的替代功能，特别是eEF1A结合和捆绑肌动蛋白的能力，使我们扩大分析，以了解这种联系的结构基础。对于酿酒酵母，eEF1a上获得的结构信息可以用于功能和调控关键残基的分子遗传分析。

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.