Jivotovskaya Antonina V, Valásek Leos, Hinnebusch Alan G, Nielsen Klaus H
Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, NIH, Building 6A/Room B1A13, Bethesda, MD 20892, USA.
Mol Cell Biol. 2006 Feb;26(4):1355-72. doi: 10.1128/MCB.26.4.1355-1372.2006.
Recruitment of the eukaryotic translation initiation factor 2 (eIF2)-GTP-Met-tRNAiMet ternary complex to the 40S ribosome is stimulated by multiple initiation factors in vitro, including eIF3, eIF1, eIF5, and eIF1A. Recruitment of mRNA is thought to require the functions of eIF4F and eIF3, with the latter serving as an adaptor between the ribosome and the 4G subunit of eIF4F. To define the factor requirements for these reactions in vivo, we examined the effects of depleting eIF2, eIF3, eIF5, or eIF4G in Saccharomyces cerevisiae cells on binding of the ternary complex, other initiation factors, and RPL41A mRNA to native 43S and 48S preinitiation complexes. Depleting eIF2, eIF3, or eIF5 reduced 40S binding of all constituents of the multifactor complex (MFC), comprised of these three factors and eIF1, supporting a mechanism of coupled 40S binding by MFC components. 40S-bound mRNA strongly accumulated in eIF5-depleted cells, even though MFC binding to 40S subunits was reduced by eIF5 depletion. Hence, stimulation of the GTPase activity of the ternary complex, a prerequisite for 60S subunit joining in vitro, is likely the rate-limiting function of eIF5 in vivo. Depleting eIF2 or eIF3 impaired mRNA binding to free 40S subunits, but depleting eIF4G led unexpectedly to accumulation of mRNA on 40S subunits. Thus, it appears that eIF3 and eIF2 are more critically required than eIF4G for stable binding of at least some mRNAs to native preinitiation complexes and that eIF4G has a rate-limiting function at a step downstream of 48S complex assembly in vivo.
在体外，真核翻译起始因子2（eIF2）-GTP-甲硫氨酰-tRNAiMet三元复合物与40S核糖体的结合受到多种起始因子的刺激，包括eIF3、eIF1、eIF5和eIF1A。mRNA的募集被认为需要eIF4F和eIF3的功能，后者作为核糖体与eIF4F的4G亚基之间的衔接子。为了确定体内这些反应所需的因子，我们研究了在酿酒酵母细胞中耗尽eIF2、eIF3、eIF5或eIF4G对三元复合物、其他起始因子以及RPL41A mRNA与天然43S和48S起始前复合物结合的影响。耗尽eIF2、eIF3或eIF5会降低由这三种因子和eIF1组成的多因子复合物（MFC）所有成分与40S的结合，这支持了MFC成分协同结合40S的机制。即使eIF5耗尽会降低MFC与40S亚基的结合，但在eIF5耗尽的细胞中，与40S结合的mRNA仍大量积累。因此，三元复合物GTPase活性的刺激是体外60S亚基加入的前提条件，这可能是eIF5在体内的限速功能。耗尽eIF2或eIF3会损害mRNA与游离40S亚基的结合，但耗尽eIF4G却意外地导致mRNA在40S亚基上积累。因此，对于至少一些mRNA与天然起始前复合物的稳定结合，eIF3和eIF2似乎比eIF4G更关键，并且eIF4G在体内48S复合物组装下游的步骤中具有限速功能。
