真核起始因子 1A 与 eIF5 之间的相互作用将 eIF1 保留在扫描起始前复合物中。
The interaction between eukaryotic initiation factor 1A and eIF5 retains eIF1 within scanning preinitiation complexes.
机构信息
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School , Boston, Massachusetts 02115, United States.
出版信息
Biochemistry. 2013 Dec 31;52(52):9510-8. doi: 10.1021/bi4009775. Epub 2013 Dec 19.
Abstract
Scanning of the mRNA transcript by the preinitiation complex (PIC) requires a panel of eukaryotic initiation factors, which includes eIF1 and eIF1A, the main transducers of stringent AUG selection. eIF1A plays an important role in start codon recognition; however, its molecular contacts with eIF5 are unknown. Using nuclear magnetic resonance, we unveil eIF1A's binding surface on the carboxyl-terminal domain of eIF5 (eIF5-CTD). We validated this interaction by observing that eIF1A does not bind to an eIF5-CTD mutant, altering the revealed eIF1A interaction site. We also found that the interaction between eIF1A and eIF5-CTD is conserved between humans and yeast. Using glutathione S-transferase pull-down assays of purified proteins, we showed that the N-terminal tail (NTT) of eIF1A mediates the interaction with eIF5-CTD and eIF1. Genetic evidence indicates that overexpressing eIF1 or eIF5 suppresses the slow growth phenotype of eIF1A-NTT mutants. These results suggest that the eIF1A-eIF5-CTD interaction during scanning PICs contributes to the maintenance of eIF1 within the open PIC.
摘要
由起始前复合物（PIC）对 mRNA 转录物的扫描需要一组真核起始因子，包括 eIF1 和 eIF1A，它们是严格 AUG 选择的主要转导因子。eIF1A 在起始密码子识别中起重要作用；然而，其与 eIF5 的分子接触尚不清楚。使用核磁共振，我们揭示了 eIF1A 在 eIF5 羧基末端结构域（eIF5-CTD）上的结合表面。我们通过观察到 eIF1A 不与改变揭示的 eIF1A 相互作用位点的 eIF5-CTD 突变体结合来验证这种相互作用。我们还发现 eIF1A 和 eIF5-CTD 之间的相互作用在人类和酵母之间是保守的。通过使用纯化蛋白的谷胱甘肽 S-转移酶下拉测定，我们表明 eIF1A 的 N 端尾巴（NTT）介导与 eIF5-CTD 和 eIF1 的相互作用。遗传证据表明，过量表达 eIF1 或 eIF5 可抑制 eIF1A-NTT 突变体的生长缓慢表型。这些结果表明，在 PIC 扫描过程中 eIF1A-eIF5-CTD 相互作用有助于维持开放 PIC 中的 eIF1。
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