Mutually cooperative binding of eukaryotic translation initiation factor (eIF) 3 and eIF4A to human eIF4G-1
Mutually cooperative binding of eukaryotic translation initiation factor (eIF) 3 and eIF4A to human eIF4G-1
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DOI：
10.1074/jbc.m007525200
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发表时间：
2000-12-29
影响因子：
4.8
通讯作者：
Rhoads, RE
中科院分区：
文献类型：
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作者：
Korneeva, NL;Lamphear, BJ;Rhoads, RE
真核翻译起始因子4G - 1（eIF4G）在mRNA募集到43S起始前复合物的过程中起着关键作用。eIF4G的中心区域结合依赖ATP的RNA解旋酶eIF4A、40S结合因子eIF3和RNA。在本研究中，我们进一步对人eIF4G中心区域的结合特性进行了表征。滴定和竞争实验均表明eIF3结合的化学计量比为1∶1。表面等离子体共振研究显示，对应于氨基酸642 - 1560、613 - 1078和975 - 1078的三个重组eIF4G片段以相似的动力学结合eIF3。由结合速率常数3.9×10⁴ M⁻¹ s⁻¹和解离速率常数1.5×10⁻³ s⁻¹推导出解离平衡常数约为42 nM。因此，eIF3结合区域包含在氨基酸残基975 - 1078内。该区域与RNA结合位点不重叠，这表明eIF3直接结合eIF4G，而不是通过RNA桥或中心的eIF4A结合位点。令人惊讶的是，eIF3和eIF4A与中心区域的结合是相互协同的；在eIF4A存在的情况下，eIF3与eIF4G的结合增加4倍，反之，在eIF3存在的情况下，eIF4A与eIF4G中心（而非羧基末端）区域的结合增加2.4倍。
Eukaryotic translation initiation factor 4G-1 (eIF4G) plays a critical role in the recruitment of mRNA to the 43 S preinitiation complex. The central region of eIF4G binds the ATP-dependent RNA helicase eIF4A, the 40 S binding factor eIF3, and RNA. In the present work, we have further characterized the binding properties of the central region of human eIF4G, Both titration and competition experiments were consistent with a 1:1 stoichiometry for eIF3 binding. Surface plasmon resonance studies showed that three recombinant eIF4G fragments corresponding to amino acids 642-1560, 613-1078, and 975-1078 bound eIF3 with similar kinetics. A dissociation equilibrium constant of similar to 42 nM was derived from an association rate constant of 3.9 x 10(4) M-1 s(-1) and dissociation rate constant of 1.5 x 10(-3) s(-1). Thus, the eIF3-binding region is included within amino acid residues 975-1078. This region does not overlap with the RNA-binding site, which suggests that eIF3 binds eIF4G directly and not through an RNA bridge, or the central eIF4A-binding site. Surprisingly, the binding of eIF3 and eIF4A to the central region was mutually cooperative; eIF3 binding to eIF4G: increased 4-fold in the presence of eIF4A, and conversely, eIF4A binding to the central (but not COOH-terminal) region of eIF4G increased 2.4-fold in the presence of eIF3.