Nielsen Klaus H, Szamecz Béla, Valásek Leos, Jivotovskaya Antonina, Shin Byung-Sik, Hinnebusch Alan G
Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, Bethesda, MD 20892-2716, USA.
EMBO J. 2004 Mar 10;23(5):1166-77. doi: 10.1038/sj.emboj.7600116. Epub 2004 Feb 19.
The binding of eIF2-GTP-tRNA(i)(Met) ternary complex (TC) to 40S subunits is impaired in yeast prt1-1 (eIF3b) mutant extracts, but evidence is lacking that TC recruitment is a critical function of eIF3 in vivo. If TC binding was rate-limiting in prt1-1 cells, overexpressing TC should suppress the temperature-sensitive phenotype and GCN4 translation should be strongly derepressed in this mutant, but neither was observed. Rather, GCN4 translation is noninducible in prt1-1 cells, and genetic analysis indicates defective ribosomal scanning between the upstream open reading frames that mediate translational control. prt1-1 cells also show reduced utilization of a near-cognate start codon, implicating eIF3 in AUG selection. Using in vivo cross-linking, we observed accumulation of TC and mRNA/eIF4G on 40S subunits and a 48S 'halfmer' in prt1-1 cells. Genetic evidence suggests that 40S-60S subunit joining is not rate-limiting in the prt1-1 mutant. Thus, eIF3b functions between 48S assembly and subunit joining to influence AUG recognition and reinitiation on GCN4 mRNA. Other mutations that disrupt eIF2-eIF3 contacts in the multifactor complex (MFC) diminished 40S-bound TC, indicating that MFC formation enhances 43S assembly in vivo.
在酵母prt1-1（eIF3b）突变体提取物中，eIF2-GTP-tRNA(i)(Met)三元复合物（TC）与40S亚基的结合受损，但缺乏证据表明TC募集是eIF3在体内的关键功能。如果在prt1-1细胞中TC结合是限速步骤，那么过表达TC应该能抑制温度敏感表型，并且该突变体中的GCN4翻译应该会强烈去抑制，但这两种情况均未观察到。相反，prt1-1细胞中的GCN4翻译是不可诱导的，并且遗传分析表明在介导翻译控制的上游开放阅读框之间存在核糖体扫描缺陷。prt1-1细胞还显示出对近同源起始密码子的利用率降低，这表明eIF3参与AUG选择。通过体内交联，我们在prt1-1细胞中观察到TC以及mRNA/eIF4G在40S亚基和48S“半体”上的积累。遗传证据表明在prt1-1突变体中40S-60S亚基结合不是限速步骤。因此，eIF3b在48S组装和亚基结合之间发挥作用，以影响GCN4 mRNA上的AUG识别和重新起始。多因子复合物（MFC）中破坏eIF2-eIF3接触的其他突变减少了与40S结合的TC，表明MFC形成在体内增强了43S组装。
