Cooperative modulation by eIF4G of eIF4E-binding to the mRNA 5′ cap in yeast involves a site partially shared by p20
Cooperative modulation by eIF4G of eIF4E-binding to the mRNA 5′ cap in yeast involves a site partially shared by p20
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DOI：
10.1093/emboj/17.16.4798
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发表时间：
1998-08-17
期刊：
影响因子：
11.4
通讯作者：
McCarthy, JEG
中科院分区：
文献类型：
--
作者：
Ptushkina, M;von der Haar, T;McCarthy, JEG
mRNA 5 ′-帽结合蛋白eIF 4 E和多接头蛋白eIF 4G之间的相互作用已经在所有的真核生物翻译组装体中得到证实。本研究利用免疫学、遗传学和生物化学方法对eIF 4 E上有助于eIF 4G结合的表面氨基酸进行了定位。帽类似物色谱和表面等离子体共振（SPR）分析表明，在这些表面区域的一类突变破坏eIF 4 E eIF 4G协会，从而多核糖体的形成和生长。野生型eIF 4 E中这些位置的残基介导eIF 4G与eIF 4 E的结合和后者的帽亲和力之间的正协同性。此外，其中两个突变赋予eIF 4G与eIF 4 E结合的温度敏感性，这与体内大量无活性核糖体80 S对的形成和细胞蛋白质合成活性的丧失相关。通过SPR估计酵母4 E结合蛋白p20对eIF 4 E的结合亲和力比eIF 4G低10倍。对第二类eIF 4 E突变的研究表明，p20仅共享eIF 4G在帽结合蛋白上的部分结合位点。这些结果为理解eIF 4 E和eIF 4G在酵母翻译中的循环方式提供了基础，并解释了p20如何作为蛋白质合成的精细调节剂而不是粗糙调节剂。
Interaction between the mRNA 5'-cap-binding protein eIF4E and the multiadaptor protein eIF4G has been demonstrated in all eukaryotic translation assemblies examined so far, This study uses immunological, genetic and biochemical methods to map the surface amino acids on eIF4E that contribute to eIF4G binding. Cap-analogue chromatography and surface plasmon resonance (SPR) analyses demonstrate that one class of mutations in these surface regions disrupts eIF4E eIF4G association, and thereby polysome formation and growth. The residues at these positions in wildtype eIF4E mediate positive cooperativity between the binding of eIF4G to eIF4E and the latter's cap-affinity. Moreover, two of the mutations confer temperature sensitivity in eIF4G binding to eIF4E which correlates with the formation of large numbers of inactive ribosome 80S couples in vivo and the loss of cellular protein synthesis activity. The yeast 4E-binding protein p20 is estimated by SPR to have a ten times lower binding affinity than eIF4G for eIF4E, Investigation of a second class of eIF4E mutations reveals that p20 shares only part of eIF4G's binding site on the cap-binding protein. The results presented provide a basis for understanding how cycling of eIF4E and eIF4G occurs in yeast translation and explains how p20 can act as a fine, but not as a coarse, regulator of protein synthesis.