Effects of poly(A)-binding protein on the interactions of translation initiation factor eIF4F and eIF4F·4B with internal ribosome entry site (IRES) of tobacco etch virus RNA

Eukaryotic Initiation Factor (eIF) 4F Binding to Barley Yellow Dwarf Virus (BYDV) 3′-Untranslated Region Correlates with Translation Efficiency

“…BTE and mutants did not interact with strong affinities with PABP, eIF4B, or eIF4A, suggesting that it is eIF4F that plays the major role in transfer of the preinitiation complex from 3Ј-to 5Ј-UTR so that translation can initiate. In contrast, eIF4F binding to PK1 RNA of tobacco etch virus is enhanced multifold by the presence of PABP (28), thus showing an alternate mechanism by which BYDV virus assembles its translation machinery. This is in agreement with an earlier predicted trend for internal ribosome entry site RNAs, which is consistent with the lower requirement for additional factors of the very well folded and stable 3Ј BTE structure compared with a single stem loop pseudoknot structure of PK1 RNA (33,39).…”

“…The binding affinity of eIF4F for 3Ј BTE did not change significantly in the presence of eIF4B (Table 1). In contrast, the eIF4F binding to tobacco etch virus pseudoknot 1 (PK1) was 4-fold stronger in the presence of both PABP and eIF4B (28). Because binding of eIF4A, eIF4B, and PABP alone was very weak, an excess of each of these proteins to eIF4F ensured that at least 90% of the eIF4F was in complex form (eIF4F⅐PABP, eIF4F⅐eIF4B, and eIF4F⅐4A) by using the concentrations predicted using the K d values (7,31) for the protein-protein interactions.…”

“…The purity of eIF4F was confirmed by 10% SDS-PAGE, and the concentration was determined using the Bradford method with BSA as a standard (27). His-tagged PABP and eIF4A protein were expressed in BL21 (DE3) pLysS Escherichia coli strain using pET19b and pET23d vector (a generous gift from Dr. D. R. Gallie, University of California, Riverside, CA), respectively, and were purified as described elsewhere (28,29).…”

Mechanism of Cytoplasmic mRNA Translation

“…Extensive biochemical and kinetic characterization of the interactions of wheat eIF4B, eIF4A, eIF4G/eIFiso4G and PABP confirm that the interactions are similar to other organisms (reviewed in Gallie, 2014;Le et al, 1997;Khan and Goss, 2005;Gallie, 2006, 2007;Cheng et al, 2008;Khan et al, 2008;Khan et al, 2009;Mayberry et al, 2009;Cheng and Gallie, 2010;Yumak et al, 2010;Khan and Goss, 2012;Cheng and Gallie, 2013). Wheat eIF4B has two tandem domains for interaction with eIF4A and PABP separated by a RNA binding domain in addition to binding domains for eIF4G/iso4G and eIF3g.…”

“…This second domain is absent in mammals and yeast PABP. PABP is also implicated in viral replication (Smith and Gray, 2010) and plant PABP was shown to interact with the reverse transcriptase of turnip mosaic virus (TuMV) (Dufresne et al, 2008) and with the 3'UTR of tobacco etch virus (TEV) to promote internal initiation (Khan et al, 2008;Khan et al, 2009;Yumak et al, 2010;Iwakawa et al, 2012).…”