The roles of individual eukaryotic translation initiation factors in ribosomal scanning and initiation codon selection
The roles of individual eukaryotic translation initiation factors in ribosomal scanning and initiation codon selection
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DOI：
10.1101/gad.1020902
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发表时间：
2002-11-15
影响因子：
10.5
通讯作者：
Kolupaeva, VG
中科院分区：
文献类型：
--
作者：
Pestova, TV;Kolupaeva, VG
为了阐明真核细胞翻译起始的机制，对完全纯化的翻译成分在体外重组的反应中确定的mRNAs上形成48S复合体进行了分析。我们发现，核糖体的40S亚基，真核细胞起始因子(EIF)3，和eIF2三级复合体形成了一个43S复合体，可以与一个非结构的5‘-非翻译区(5’-UTR)的5‘端结合，在CIF1的存在下，扫描并定位起始密码子，而不需要三磷酸腺苷(ATP)或与ATP水解相关的因子(eIF4A，eIF4B，eIF4F)。ATP、EIFS4A和4B以及cIF4F的eIF4G亚基的中央结构域增强了扫描油的非结构5‘-UTRs。它们的遗漏增加了扫描对EIFS I和1A的依赖性。含有弱二级结构的核糖体运动油5‘-UTRs需要ATP和RNA解旋酶。EIF4F对于扫描是必不可少的，在没有eIF4F的情况下，EIFS4A和4B不足以促进这一进程。我们报道，eIF1除了在扫描中起作用外，还在起始密码子选择中起主要作用。在没有eIF1的情况下，43S复合体不再能够区分同源和非同源起始密码子，也不能感知起始密码子的核苷酸上下文，而是能够在5‘-近端的Aug三联体上组装48S复合体，该三联体距离mRNA的5’端只有1、2和4个核苷酸。
To elucidate an outline of the mechanism of eukaryotic translation initiation, 48S complex formation was analyzed on defined mRNAs in reactions reconstituted in vitro from fully purified translation components. We found that a ribosomal 40S subunit, eukaryotic initiation factor (eIF) 3, and the eIF2 tertiary complex form a 43S complex that can bind to the 5'-end of an unstructured 5'-untranslated region (5'-UTR) and in the presence of cIF1 scan along it and locate the initiation codon without a requirement for adenosine triphosphate (ATP) or factors (eIF4A, eIF4B, eIF4F) associated with ATP hydrolysis. Scanning oil unstructured 5'-UTRs was enhanced by ATP, eIFs 4A and 4B, and the central domain of the eIF4G subunit of cIF4F. Their omission increased the dependence of scanning on eIFs I and 1A. Ribosomal movement oil 5'-UTRs containing even weak secondary structures required ATP and RNA helicases. eIF4F was essential for scanning, and eIFs 4A and 4B were insufficient to promote this process in the absence of eIF4F. We report that in addition to its function in scanning, eIF1 also plays a principal role in initiation codon selection. In the absence of eIF1, 43S complexes could no longer discriminate between cognate and noncognate initiation codons or sense the nucleotide context of initiation codons and were able to assemble 48S complexes on 5'-proximal AUG triplets located only 1, 2, and 4 nt from the 5'-end of mRNA.