Translation initiation at non-AUG codons mediated by weakened association of eukaryotic initiation factor (eIF) 2 subunits
Translation initiation at non-AUG codons mediated by weakened association of eukaryotic initiation factor (eIF) 2 subunits
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DOI：
10.1042/bj20020556
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发表时间：
2002-10-15
影响因子：
4.1
通讯作者：
Castilho, BA
中科院分区：
文献类型：
--
作者：
Hashimoto, NN;Carnevalli, LS;Castilho, BA
异源三聚体真核起始因子（eIF）2以GTP依赖性模式结合起始剂甲硫氨酰-tRNA并将其递送至40 S核糖体亚基。在本研究中，我们已经确定了氨基酸残基eIF 2 β所需的结合eIF 2 γ在酵母中。在eIF 2 β的中心区域的6个残基的改变废除了这种相互作用，如GST-下拉和双杂交测定，并导致细胞致死。用丙氨酸残基取代（131）Tyr和（132）Ser（（YS）-Y-131），尽管在这些测定中消除了与eIF 2 gamma的结合，但在体内产生了功能性但有缺陷的蛋白质，赋予细胞温度敏感的生长表型。通过免疫共沉淀显示，这种突变蛋白与eIF 2 γ在体内的关联显著减弱。eIF 2 β中的（YS）-Y-131突变允许翻译在非AUG密码子处起始，如通过在HIS 4 mRNA中携带起始密码子突变的细胞在不存在组氨酸的情况下生长的能力所定义的。该突变与（254）Ser -> Tyr改变（先前分离的起始密码子突变的抑制因子，其已显示增加三元复合物中的自发GTP水解）的组合引起隐性致死性，表明加性缺陷。因此，这两个亚基的相互作用受损代表了eIF 2功能的一种新型缺陷，提供了体内证据，证明eIF 2 β和eIF 2 γ之间的相互作用强度决定了AUG密码子在翻译起始中的正确使用。
The heterotrimeric eukaryotic initiation factor (eIF) 2 binds the initiator methionyl-tRNA in a GTP-dependent mode and delivers it to the 40 S ribosomal subunit. In the present study, we have identified amino acid residues in eIF2beta required for binding to eIF2gamma in yeast. Alteration of six residues in the central region of eIF2beta abolished this interaction, as determined by GST-pull down and two-hybrid assays, and leads to cell lethality. Substitution of (131)Tyr and (132)Ser by alanine residues ((YS)-Y-131), although abolishing the binding to eIF2gamma in these assays, resulted in a functional but defective protein in vivo, imparting a temperature-sensitive growth phenotype to cells. A dramatically weakened association of this mutant protein with eIF2gamma in vivo was shown by co-immunoprecipitation. The (YS)-Y-131 mutation in eIF2beta allows translation to initiate at non-AUG codons, as defined by the ability of cells carrying an initiator codon mutation in the HIS4 mRNA to grow in the absence of histidine. The combination of this mutation with the (254)Ser --> Tyr alteration, a previously isolated suppressor of initiator codon mutations which has been shown to increase the spontaneous GTP hydrolysis in the ternary complex, caused a recessive lethality, suggesting additive defects. Thus the impaired interaction of these two subunits represents a novel type of defect in eIF2 function, providing in vivo evidence that the strength of interaction between eIF2beta and eIF2gamma defines the correct usage of the AUG codon for translation initiation.