eIF2A是第一个被发现的真核细胞起始tRNA携带者，但其确切功能至今仍然神秘。与翻译起始因子的特点不同，据报道，eIF2A在多种人类癌细胞系中被发现为非细胞质蛋白。由于无法获得高产量的可溶性重组蛋白，研究eIF2A的机制一直受到限制。在本研究中，我们开发了一种纯化策略，从大肠杆菌和昆虫细胞中获得约360倍和约6000倍于先前报道的人重组eIF2A。使用哺乳动物体外翻译系统，我们发现增加重组人eIF2A的水平会抑制多个报告基因mRNA的翻译，包括通过恰当和近似起始密码子翻译的mRNA，并且是在起始密码子识别之前发生的。eIF2A还抑制了由四种类型的无帽病毒IRESs引导的翻译，包括不需要起始因子或起始tRNA的CrPVIGRIRES，这表明过量的eIF2A会束缚40S亚单位。补充额外的40S亚单位可以防止eIF2A介导的抑制作用，并且拉脱实验证明了重组eIF2A与纯化的40S亚单位之间的直接结合。这些数据支持一种模型，即必须将eIF2A与翻译机器隔离开来，以避免包结40S核糖体亚单位。© 2023年该作者。由牛津大学出版社代表核酸研究出版。

eIF2A was the first eukaryotic initiator tRNA carrier discovered but its exact function has remained enigmatic. Uncharacteristic of translation initiation factors, eIF2A is reported to be non-cytosolic in multiple human cancer cell lines. Attempts to study eIF2A mechanistically have been limited by the inability to achieve high yield of soluble recombinant protein. Here, we developed a purification paradigm that yields ∼360-fold and ∼6000-fold more recombinant human eIF2A from Escherichia coli and insect cells, respectively, than previous reports. Using a mammalian in vitro translation system, we found that increased levels of recombinant human eIF2A inhibit translation of multiple reporter mRNAs, including those that are translated by cognate and near-cognate start codons, and does so prior to start codon recognition. eIF2A also inhibited translation directed by all four types of cap-independent viral IRESs, including the CrPV IGR IRES that does not require initiation factors or initiator tRNA, suggesting excess eIF2A sequesters 40S subunits. Supplementation with additional 40S subunits prevented eIF2A-mediated inhibition and pull-down assays demonstrated direct binding between recombinant eIF2A and purified 40S subunits. These data support a model that eIF2A must be kept away from the translation machinery to avoid sequestering 40S ribosomal subunits.© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.