Paek Ki Young, Hong Ka Young, Ryu Incheol, Park Sung Mi, Keum Sun Ju, Kwon Oh Sung, Jang Sung Key
POSTECH Biotechnology Center, Department of Life Sciences, and.
POSTECH Biotechnology Center, Department of Life Sciences, and Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, Kyungbuk 790-784, Korea
Proc Natl Acad Sci U S A. 2015 Jan 27;112(4):1041-6. doi: 10.1073/pnas.1416883112. Epub 2015 Jan 12.
Eukaryotic translation initiation commences at the initiation codon near the 5' end of mRNA by a 40S ribosomal subunit, and the recruitment of a 40S ribosome to an mRNA is facilitated by translation initiation factors interacting with the m(7)G cap and/or poly(A) tail. The 40S ribosome recruited to an mRNA is then transferred to the AUG initiation codon with the help of translation initiation factors. To understand the mechanism by which the ribosome finds an initiation codon, we investigated the role of eIF4G in finding the translational initiation codon. An artificial polypeptide eIF4G fused with MS2 was localized downstream of the reporter gene through MS2-binding sites inserted in the 3' UTR of the mRNA. Translation of the reporter was greatly enhanced by the eIF4G-MS2 fusion protein regardless of the presence of a cap structure. Moreover, eIF4G-MS2 tethered at the 3' UTR enhanced translation of the second cistron of a dicistronic mRNA. The encephalomyocarditis virus internal ribosome entry site, a natural translational-enhancing element facilitating translation through an interaction with eIF4G, positioned downstream of a reporter gene, also enhanced translation of the upstream gene in a cap-independent manner. Finally, we mathematically modeled the effect of distance between the cap structure and initiation codon on the translation efficiency of mRNAs. The most plausible explanation for translational enhancement by the translational-enhancing sites is recognition of the initiation codon by the ribosome bound to the ribosome-recruiting sites through "RNA looping." The RNA looping hypothesis provides a logical explanation for augmentation of translation by enhancing elements located upstream and/or downstream of a protein-coding region.
真核生物的翻译起始由40S核糖体亚基在mRNA 5'端附近的起始密码子处开始，翻译起始因子与m(7)G帽和/或多聚(A)尾相互作用，促进40S核糖体与mRNA的结合。然后，在翻译起始因子的帮助下，募集到mRNA上的40S核糖体转移至AUG起始密码子。为了解核糖体找到起始密码子的机制，我们研究了真核起始因子4G（eIF4G）在寻找翻译起始密码子中的作用。通过插入mRNA 3'非翻译区（UTR）的MS2结合位点，将与MS2融合的人工多肽eIF4G定位在报告基因的下游。无论是否存在帽结构，eIF4G-MS2融合蛋白都能极大地增强报告基因的翻译。此外，拴系在3'UTR处的eIF4G-MS2增强了双顺反子mRNA第二个顺反子的翻译。脑心肌炎病毒内部核糖体进入位点是一种天然的翻译增强元件，通过与eIF4G相互作用促进翻译，该元件位于报告基因下游，也能以不依赖帽的方式增强上游基因的翻译。最后，我们对帽结构与起始密码子之间的距离对mRNA翻译效率的影响进行了数学建模。翻译增强位点促进翻译最合理的解释是，通过“RNA环化”，与核糖体募集位点结合的核糖体识别起始密码子。RNA环化假说为蛋白质编码区上游和/或下游的增强元件增强翻译提供了一个合理的解释。
