Development and characterization of a reconstituted yeast translation initiation system
Development and characterization of a reconstituted yeast translation initiation system
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DOI：
10.1017/s1355838202029527
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发表时间：
2002-03-01
期刊：
影响因子：
4.5
通讯作者：
Lorsch, JR
中科院分区：
文献类型：
--
作者：
Algire, MA;Maag, D;Lorsch, JR
为了在真核生物翻译起始的体内和体外研究之间搭建一座桥梁，我们利用来自酿酒酵母（Saccharomyces cerevisiae）的组分开发了一种重组翻译起始系统。我们纯化了一组最少的起始因子（eIFs），它们与酵母80S核糖体、GTP以及起始甲硫氨酰 - tRNA一起，足以在一个最简mRNA模板上组装有活性的起始复合物。我们已经探究了起始复合物组装途径中各个步骤以及体外第一个肽键形成的动力学。在这个系统中，有活性的起始复合物的形成依赖于核糖体、mRNA、Met - tRNA（i）、GTP水解、eIF1、eIF1A、eIF2、eIF5和eIF5B。我们的数据表明，eIF1和eIF1A都促进eIF2·GTP·Met - tRNA（i）复合物与40S核糖体亚基的结合以形成43S复合物。eIF5在43S复合物形成之后的一个步骤起刺激作用，这与其在起始密码子识别时激活eIF2的GTP水解的假定作用是一致的。eIF5B的存在是40S和60S亚基结合形成80S起始复合物所必需的。在这个重组系统中，这些因子各自作用的步骤与先前体内研究以及使用重组哺乳动物系统的研究数据是一致的，这表明该系统重现了真核细胞翻译起始中的基本事件。这个系统应该能使我们将强大的酵母遗传学和分子生物学实验与体外动力学和生物物理学实验相结合，从而更好地理解这个核心而复杂的过程的分子机制。
To provide a bridge between in vivo and in vitro studies of eukaryotic translation initiation, we have developed a reconstituted translation initiation system using components from the yeast Saccharomyces cerevisiae. We have purified a minimal set of initiation factors (eIFs) that, together with yeast 80S ribosomes, GTP, and initiator methionyl-tRNA, are sufficient to assemble active initiation complexes on a minimal mRNA template. The kinetics of various steps in the pathway of initiation complex assembly and the formation of the first peptide bond in vitro have been explored. The formation of active initiation complexes in this system is dependent on ribosomes, mRNA, Met-tRNA(i), GTP hydrolysis, eIF1, eIF1 A, eIF2, eIF5, and eIF5B. Our data indicate that eIF1 and eIF1 A both facilitate the binding of the eIF2.GTP.Met-tRNA(i) complex to the 40S ribosomal subunit to form the 43S complex. eIF5 stimulates a step after 43S complex formation, consistent with its proposed role in activating GTP hydrolysis by eIF2 upon initiation codon recognition. The presence of eIF5B is required for the joining of the 40S and 60S subunits to form the 80S initiation complex. The step at which each of these factors acts in this reconstituted system is in agreement with previous data from in vivo studies and work using reconstituted mammalian systems, indicating that the system recapitulates fundamental events in translation initiation in eukaryotic cells. This system should allow us to couple powerful yeast genetic and molecular biological experiments with in vitro kinetic and biophysical experiments, yielding a better understanding of the molecular mechanics of this central, complex process.