From the Department of Chemistry, Hunter College and the Graduate Center of the City University of New York, New York, New York 10065.
J Biol Chem. 2014 Feb 14;289(7):4286-94. doi: 10.1074/jbc.M113.530329. Epub 2013 Dec 30.
Eukaryotic initiation factor (eIF) 4F binding to mRNA is the first committed step in cap-dependent protein synthesis. Barley yellow dwarf virus (BYDV) employs a cap-independent mechanism of translation initiation that is mediated by a structural BYDV translation element (BTE) located in the 3'-UTR of its mRNA. eIF4F bound the BTE and a translationally inactive mutant with high affinity, thus questioning the role of eIF4F in translation of BYDV. To examine the effects of eIF4F in BYDV translation initiation, BTE mutants with widely different in vitro translation efficiencies ranging from 5 to 164% compared with WT were studied. Using fluorescence anisotropy to obtain quantitative data, we show 1) the equilibrium binding affinity (complex stability) correlated well with translation efficiency, whereas the "on" rate of binding did not; 2) other unidentified proteins or small molecules in wheat germ extract prevented eIF4F binding to mutant BTE but not WT BTE; 3) BTE mutant-eIF4F interactions were found to be both enthalpically and entropically favorable with an enthalpic contribution of 52-90% to ΔG° at 25 °C, suggesting that hydrogen bonding contributes to stability; and 4) in contrast to cap-dependent and tobacco etch virus internal ribosome entry site interaction with eIF4F, poly(A)-binding protein did not increase eIF4F binding. Further, the eIF4F bound to the 3' BTE with higher affinity than for either m(7)G cap or tobacco etch virus internal ribosome entry site, suggesting that the 3' BTE may play a role in sequestering host cell initiation factors and possibly regulating the switch from replication to translation.
真核起始因子 (eIF) 4F 与 mRNA 的结合是帽依赖性蛋白合成的第一步。大麦黄花叶病毒 (BYDV) 采用一种非依赖帽的翻译起始机制，该机制由其 mRNA 3'UTR 中的结构 BYDV 翻译元件 (BTE) 介导。eIF4F 与 BTE 及其具有高亲和力的翻译失活突变体结合，从而对 eIF4F 在 BYDV 翻译中的作用提出质疑。为了研究 eIF4F 在 BYDV 翻译起始中的作用，研究了与 WT 相比体外翻译效率差异很大（5%至 164%）的 BTE 突变体。通过荧光各向异性获得定量数据，我们发现：1）平衡结合亲和力（复合物稳定性）与翻译效率密切相关，而结合的“开启”速率则不然；2）小麦胚提取物中的其他未鉴定蛋白或小分子阻止 eIF4F 与突变 BTE 结合，但不阻止 WT BTE 结合；3）发现 BTE 突变体-eIF4F 相互作用在热力学上是有利的，在 25°C 时，焓变对 ΔG°的贡献为 52%-90%，表明氢键有助于稳定性；4）与帽依赖性和烟草蚀纹病毒内部核糖体进入位点与 eIF4F 的相互作用不同，多聚(A)结合蛋白不会增加 eIF4F 结合。此外，eIF4F 与 3' BTE 的结合亲和力高于 m(7)G 帽或烟草蚀纹病毒内部核糖体进入位点，这表明 3' BTE 可能在隔离宿主细胞起始因子并可能调节从复制到翻译的转换方面发挥作用。
