Algire Mikkel A, Maag David, Savio Peter, Acker Michael G, Tarun Salvador Z, Sachs Alan B, Asano Katsura, Nielsen Klaus H, Olsen Deanne S, Phan Lon, Hinnebusch Alan G, Lorsch Jon R
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185, USA.
RNA. 2002 Mar;8(3):382-97. doi: 10.1017/s1355838202029527.
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 (elFs) 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-tRNAi, GTP hydrolysis, elF1, elF1A, elF2, elF5, and elF5B. Our data indicate that elF1 and elF1A both facilitate the binding of the elF2 x GTP x Met-tRNAi complex to the 40S ribosomal subunit to form the 43S complex. elF5 stimulates a step after 43S complex formation, consistent with its proposed role in activating GTP hydrolysis by elF2 upon initiation codon recognition. The presence of elF5B 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.
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，我们利用酿酒酵母的成分开发了一种重构的翻译起始系统。我们已经纯化了一组最小的起始因子（elFs），这些因子与酵母80S核糖体、GTP和起始甲硫氨酰 - tRNA一起，足以在最小的mRNA模板上组装活性起始复合物。我们已经探索了起始复合物组装途径中各个步骤的动力学以及体外第一个肽键的形成。该系统中活性起始复合物的形成依赖于核糖体、mRNA、Met - tRNAi、GTP水解、elF1、elF1A、elF2、elF5和elF5B。我们的数据表明，elF1和elF1A都促进elF2·GTP·Met - tRNAi复合物与40S核糖体亚基的结合，以形成43S复合物。elF5在43S复合物形成后刺激一个步骤，这与其在起始密码子识别时激活elF2的GTP水解的假定作用一致。40S和60S亚基结合形成80S起始复合物需要elF5B的存在。这些因子在这个重构系统中发挥作用的步骤与先前体内研究以及使用重构哺乳动物系统的工作数据一致，表明该系统概括了真核细胞翻译起始中的基本事件。这个系统应该使我们能够将强大的酵母遗传和分子生物学实验与体外动力学和生物物理实验相结合，从而更好地理解这个核心复杂过程的分子机制。
