Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Structure. 2013 Dec 3;21(12):2197-207. doi: 10.1016/j.str.2013.09.016. Epub 2013 Oct 31.
Recognition of the mRNA 5' m⁷G(5')ppp(5')N cap is key to translation initiation for most eukaryotic mRNAs. The cap is bound by the eIF4F complex, consisting of a cap-binding protein (eIF4E), a "scaffold" protein (eIF4G), and an RNA helicase (eIF4A). As a central early step in initiation, regulation of eIF4F is crucial for cellular viability. Although the structure and function of eIF4E have been defined, a dynamic mechanistic picture of its activity at the molecular level in the eIF4F·mRNA complex is still unavailable. Here, using single-molecule fluorescence, we measured the effects of Saccharomyces cerevisiae eIF4F factors, mRNA secondary structure, and the poly(A)-binding protein Pab1p on eIF4E-mRNA binding dynamics. Our data provide an integrated picture of how eIF4G and mRNA structure modulate eIF4E-mRNA interaction, and uncover an eIF4G- and poly(A)-independent activity of poly(A)-binding protein that prolongs the eIF4E·mRNA complex lifetime.
真核生物大多数 mRNA 的翻译起始都依赖于对 mRNA 5' 帽结构 m⁷G(5')ppp(5')N 的识别。帽结构由 eIF4F 复合物结合，该复合物由一个帽结合蛋白（eIF4E）、一个“支架”蛋白（eIF4G）和一个 RNA 解旋酶（eIF4A）组成。作为起始的一个关键早期步骤，eIF4F 的调节对于细胞活力至关重要。尽管 eIF4E 的结构和功能已经确定，但 eIF4F·mRNA 复合物中其在分子水平上的活性的动态机制图景仍然难以获得。在这里，我们使用单分子荧光技术，测量了酿酒酵母 eIF4F 因子、mRNA 二级结构和 poly(A)-结合蛋白 Pab1p 对 eIF4E-mRNA 结合动力学的影响。我们的数据提供了一个综合的图景，说明了 eIF4G 和 mRNA 结构如何调节 eIF4E-mRNA 相互作用，并揭示了 poly(A)-结合蛋白的一种 eIF4G 和 poly(A) 非依赖性活性，该活性延长了 eIF4E·mRNA 复合物的寿命。
