Khan Mateen A, Goss Dixie J
Department of Chemistry, Hunter College and Graduate Center of the City University of New York, New York, New York 10021, USA.
Biochemistry. 2005 Mar 22;44(11):4510-6. doi: 10.1021/bi047298g.
Previous kinetic binding studies of wheat germ protein synthesis eukaryotic translational initiation factor eIFiso4F and its subunit, eIFiso4E, with m(7)GTP and mRNA analogues indicated that binding occurred by a two-step process with the first step occurring at a rate close to the diffusion-controlled rate [Sha, M., Wang, Y., Xiang, T., van Heerden, A., Browning, K. S., and Goss, D. J. (1995) J. Biol. Chem. 270, 29904-29909]. The kinetic effects of eIF4B, PABP, and wheat germ eIFiso4F with two mRNA cap analogues and the temperature dependence of this reaction were measured and compared. The Arrhenius activation energies for binding of the two mRNA cap analogues, Ant-m(7)GTP and m(7)GpppG, were significantly different. Fluorescence stopped-flow studies of the eIFiso4F.eIF4B protein complex with two m(7)G cap analogues show a concentration-independent conformational change. The rate of this conformational change was approximately 2.4-fold faster for the eIFiso4F.eIF4B complex compared with our previous studies of eIFiso4F [Sha, M., Wang, Y., Xiang, T., van Heerden, A., Browning, K. S., and Goss, D. J. (1995) J. Biol. Chem. 270, 29904-29909]. The dissociation rates were 3.7- and 5.4-fold slower for eIFiso4F.Ant-m(7)GTP and eIFiso4F.m(7)GpppG, respectively, in the presence of eIF4B and PABP. These studies show that eIF4B and PABP enhance the interaction with the cap and probably are involved in protein-protein interactions as well. The temperature dependence of the cap binding reaction was markedly reduced in the presence of either eIF4B or PABP. However, when both eIF4B and PABP were present, not only was the energy barrier reduced but the binding rate was faster. Since cap binding is thought to be the rate-limiting step in protein synthesis, these two proteins may perform a critical function in regulation of the overall protein synthesis efficiency. This suggests that the presence of both proteins leads to a rapid, stable complex, which serves as a scaffold for further initiation complex formation.
此前关于小麦胚芽蛋白合成真核翻译起始因子eIFiso4F及其亚基eIFiso4E与m⁷GTP和mRNA类似物的动力学结合研究表明，结合过程分两步进行，第一步的发生速率接近扩散控制速率[Sha, M., Wang, Y., Xiang, T., van Heerden, A., Browning, K. S., and Goss, D. J. (1995) J. Biol. Chem. 270, 29904 - 29909]。测定并比较了eIF4B、PABP和小麦胚芽eIFiso4F与两种mRNA帽类似物的动力学效应以及该反应的温度依赖性。两种mRNA帽类似物Ant - m⁷GTP和m⁷GpppG结合的阿仑尼乌斯活化能显著不同。对eIFiso4F.eIF4B蛋白复合物与两种m⁷G帽类似物的荧光停流研究显示出一种浓度无关的构象变化。与我们之前对eIFiso4F的研究[Sha, M., Wang, Y., Xiang, T., van Heerden, A., Browning, K. S., and Goss, D. J. (1995) J. Biol. Chem. 270, 29904 - 29909]相比，eIFiso4F.eIF4B复合物的这种构象变化速率快约2.4倍。在存在eIF4B和PABP的情况下，eIFiso4F.Ant - m⁷GTP和eIFiso4F.m⁷GpppG的解离速率分别慢3.7倍和5.4倍。这些研究表明，eIF4B和PABP增强了与帽的相互作用，并且可能也参与了蛋白质 - 蛋白质相互作用。在存在eIF4B或PABP的情况下，帽结合反应的温度依赖性显著降低。然而，当同时存在eIF4B和PABP时，不仅能量障碍降低，而且结合速率更快。由于帽结合被认为是蛋白质合成中的限速步骤，这两种蛋白质可能在调节整体蛋白质合成效率方面发挥关键作用。这表明这两种蛋白质的存在导致形成一种快速、稳定的复合物，该复合物作为进一步起始复合物形成的支架。
