Journal
BIOCHIMIE
Volume 88, Issue 3-4, Pages 329-340Publisher
ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER
DOI: 10.1016/j.biochi.2005.09.002
Keywords
turnip mosaic virus; genome-linked protein; eukaryotic initiation factor; translational control; surface plasmon resonance
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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.
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