Regulators of Translation Elongation Factor eEF1A

描述（由申请人提供）：真核翻译延伸因子（eEFs）是蛋白质合成准确性和效率的决定因素。越来越多的证据也支持翻译延伸在基因表达调控中的重要性。在真菌中的三种eEFs中，eEF1A是GTP结合蛋白，可将氨基酰trna （aa-tRNA）传递到核糖体a位点。很明显，eEF1A在物理和功能上与其他辅助因子，如鸟嘌呤核苷酸交换因子，以及蛋白质和翻译延伸外的过程相互作用。eEF1A活性或水平的改变与重要的细胞表型有关，因为该蛋白在转化细胞系和某些癌症中过度表达，表达增加导致易感性转化。此外，最近发现的两种化合物可靶向eEF1A的肌动蛋白捆绑活性，影响男性生殖系统并抑制黑色素瘤细胞的生长。该提案的统一主题是eEF1A的调控影响翻译伸长的效率、准确性和控制。这种调节可能通过与辅助因子如aa-tRNA和actin的相互作用或激酶的修饰而发生。提出的三个目标打破了围绕询问这些关键相互作用和功能的突变类的eEF1A的机制分析。Aim 1利用影响eEF1A多种活性的eEF1A突变体来解决eEF1A活性改变影响蛋白质合成起始步骤的机制，关键调控起始因子eIF2的磷酸化证明了这一点。结合对影响翻译保真度和核苷酸结合的突变的分析，这些研究将为翻译的多个方面如何联系，eEF1A活性与其他细胞过程之间的机制和信号，以及eEF1A改变如何影响延伸提供新的见解。目的2通过磷酸化位点分析翻译延伸和eEF1A活性的调控。最近的高通量质谱数据为解决这个问题提供了丰富的资源，我们的初步结果显示了这些位点在体内的关键作用。Aim 3利用最先进的技术，通过深度测序方法观察所有核糖体相关mrna的状态，以了解eEF1A关键活性改变对全局基因表达的重叠效应。这一建议反映了需要在细胞过程之间进行思考，并将与全局方法协调，从翻译的角度确定eEF1A活性改变的影响的广度和特异性。

DESCRIPTION (provided by applicant): The eukaryotic translation Elongation Factors (eEFs) are determinants of the accuracy and efficiency of protein synthesis. A growing body of evidence also supports the importance of translation elongation in the regulation of gene expression. Of the three eEFs in fungi, eEF1A is the GTP binding protein that delivers aminoacyl-tRNA (aa-tRNA) to the ribosomal A-site. It has become clear that eEF1A physically and functionally interacts with other cofactors such as a guanine nucleotide exchange factor, as well as proteins and processes outside translation elongation. Altered activity or levels of eEF1A are linked to important cellular phenotypes, as the protein is overexpressed in transformed cell lines and some cancers and increased expression results in susceptibility to transformation. Furthermore, two compounds have been recently identified that target the actin bundling activity of eEF1A, affecting the male reproduction system and inhibiting the growth of melanoma cells. The unifying theme of this proposal is that regulation of eEF1A affects the efficiency, accuracy and control of translation elongation. This regulation may occur through interactions with cofactors such as aa-tRNA and actin or modification by kinases. The three aims proposed break down the mechanistic analysis of eEF1A around classes of mutants that interrogate these key interactions and functions. Aim 1 utilizes eEF1A mutants that affect the multiple activities of eEF1A to address the mechanism by which altered eEF1A activity affects the initiation step of protein synthesis as evidenced by phosphorylation of a key regulatory initiation factor, eIF2. Linked with analysis of mutations that affect translational fidelity and nucleotide binding, these studies will provide new insights into how multiple aspects of translation may be linked, the mechanism and signal between activities of eEF1A and other cellular process, and how eEF1A alterations affect elongation. Aim 2 provides an analysis of the regulation of translation elongation and eEF1A activity via sites of phosphorylation. Recent high throughput MS data has provided a rich resource to address this question, and our preliminary results show the key role of these sites in vivo. Aim 3 utilizes state of the art technology to look at the status of all ribosome associated mRNAs with deep sequencing methods to understand the overlapping effects of alterations in the key activities of eEF1A on global gene expression. This proposal reflects the need to think between cellular processes, and will coordinate directed with global approaches to determine the breadth and specificity of the effects of altered activities of eEF1A from the perspective of translation.