Binding analyses for the interaction between plant virus genome-linked protein (VPg) and plant translational initiation factors
Binding analyses for the interaction between plant virus genome-linked protein (VPg) and plant translational initiation factors
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DOI：
10.1016/j.biochi.2005.09.002
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发表时间：
2006-03-01
期刊：
影响因子：
3.9
通讯作者：
Natsuaki, T
中科院分区：
文献类型：
--
作者：
Miyoshi, H;Suehiro, N;Natsuaki, T
表达和纯化了芜菁花叶病毒(TuMV)、基因组连锁蛋白(VPG)和拟南芥翻译起始因子，研究了它们的结合特性和动力学。1117GTP-Sephose柱亲和层析表明，粗制TuMV VPG能洗脱结合的拟南风EIF(Iso)4E。进一步用纯化的VPG和其他A.thaliana eIF4E亚型进行的柱研究表明，VPG优先结合eIF(Iso)4E。结构数据表明，EIF(Iso)4E中的Trp-46和Trp-92与帽子识别有关。当Trp46或Trp-92改为Leu时。EIF(Iso)4E失去了与VPG和m(7)GTP-Sepharose形成络合物的能力。这表明VPG结合位点位于EIF(Iso)4E上的帽识别口袋内或附近。用表面等离子体共振(SPR)技术测定了VPG和封端RNA低聚物与ELF(Iso)4E相互作用的亲和常数。K-D值表明，VPG与eIF(Iso)4E的结合亲和力强于封端RNA。这表明病毒VPG可以通过隔离eIF(Iso)4E来干扰寄主植物细胞mRNA上翻译起始复合体的形成。进一步的亲和层析实验表明，VPG与eIF(Iso)4E和eIF(Iso)4G形成三元络合物。因此。VPG可能通过作为另一种帽状结构参与病毒的翻译起始。(C)2005年爱思唯尔集团。版权所有。
The turnip mosaic virus (TuMV) genome-linked protein (VPg) and Arabidopsis thaliana translation initiation factors were expressed and purified in order to investigate their binding properties and kinetics. Affinity chromatography on 1117 GTP-sepharose showed that bound A. thaliana eIF(iso)4E was eluted with crude TuMV VPg. Further column studies with purified VPg and other A. thaliana eIF4E isoforms showed that VPg preferentially bound eIF(iso)4E. Structural data implicate Trp-46 and Trp-92 in eIF(iso)4E in cap recognition. When Trp46 or Trp-92 were changed to Leu. eIF(iso)4E lost the ability to form a complex with both VPg and m(7) GTP-sepharose. This suggests that the VPg-binding site is located in or near the cap-recognition pocket on eIF(iso)4E. Affinity constants for the interactions with elF(iso)4E of VPg and capped RNA oligomer were determined using surface plasmon resonance (SPR). The K-D values showed that the binging affinity of VPg for eIF(iso)4E is stronger than that of capped RNA. This suggests that viral VPg can interfere with formation of a translational initiation complex on host plant cellular mRNA by sequestering eIF(iso)4E. Further experiments with affinity chromatography showed that VPg forms a ternary complex with eIF(iso)4E and eIF(iso)4G. Thus. VPg may participate in viral translational initiation by functioning as an alternative cap-like structure. (c) 2005 Elsevier SAS. All rights reserved.