Chaudhuri J, Chowdhury D, Maitra U
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine of Yeshiva University, Jack and Pearl Resnick Campus, Bronx, New York 10461, USA.
J Biol Chem. 1999 Jun 18;274(25):17975-80. doi: 10.1074/jbc.274.25.17975.
We have used an in vitro translation initiation assay to investigate the requirements for the efficient transfer of Met-tRNAf (as Met-tRNAf.eIF2.GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA (or an AUG codon) to form the 40 S preinitiation complex. We observed that the 17-kDa initiation factor eIF1A is necessary and sufficient to mediate nearly quantitative transfer of Met-tRNAf to isolated 40 S ribosomal subunits. However, the addition of 60 S ribosomal subunits to the 40 S preinitiation complex formed under these conditions disrupted the 40 S complex resulting in dissociation of Met-tRNAf from the 40 S subunit. When the eIF1A-dependent preinitiation reaction was carried out with 40 S ribosomal subunits that had been preincubated with eIF3, the 40 S preinitiation complex formed included bound eIF3 (40 S.eIF3. Met-tRNAf.eIF2.GTP). In contrast to the complex lacking eIF3, this complex was not disrupted by the addition of 60 S ribosomal subunits. These results suggest that in vivo, both eIF1A and eIF3 are required to form a stable 40 S preinitiation complex, eIF1A catalyzing the transfer of Met-tRNAf.eIF2.GTP to 40 S subunits, and eIF3 stabilizing the resulting complex and preventing its disruption by 60 S ribosomal subunits.
我们使用了一种体外翻译起始测定法，以研究在没有mRNA（或AUG密码子）的情况下，将甲硫氨酰 - tRNAf（作为甲硫氨酰 - tRNAf·eIF2·GTP三元复合物）有效转移至40S核糖体亚基以形成40S起始前复合物的条件。我们观察到，17 kDa的起始因子eIF1A对于介导甲硫氨酰 - tRNAf几乎定量转移至分离的40S核糖体亚基是必要且充分的。然而，在这些条件下形成的40S起始前复合物中加入60S核糖体亚基会破坏40S复合物，导致甲硫氨酰 - tRNAf从40S亚基上解离。当使用预先与eIF3一起预孵育的40S核糖体亚基进行依赖eIF1A的起始前反应时，形成的40S起始前复合物包含结合的eIF3（40S·eIF3·甲硫氨酰 - tRNAf·eIF2·GTP）。与缺乏eIF3的复合物相反，该复合物不会因加入60S核糖体亚基而被破坏。这些结果表明，在体内，eIF1A和eIF3都是形成稳定的40S起始前复合物所必需的，eIF1A催化甲硫氨酰 - tRNAf·eIF2·GTP转移至40S亚基，而eIF3稳定所得复合物并防止其被60S核糖体亚基破坏。
